ALBERT

Description: Affinity of platelet integrin αIIbβ3 for ligands is dynamically regulated and the activation of αIIbβ3 has been analyzed by binding assays using fibrinogen or ligand mimetic antibodies such as PAC1. Ligand binding assays are suitable for the condition when the number of receptors would reach a plateau phase before the end of incubation period. However, these assays would not give us useful information when the receptors are exposed only transiently followed by rapid inactivation within a few minutes. We found transient aggregation of platelets from a P2Y12-deficient patient (OSP-1) when platelets were stimulated with PAR1-activating peptide (PAR1-AP), PAR4-AP or U46619. Therefore, we hypothesized that transient αIIbβ3 activation occurs under P2Y12-deficient conditions. To estimate time-dependent changes of activated αIIbβ3, we analyzed PAC1 binding rates by measuring PAC1 binding every 30 sec (or 1 min) after the addition of PAC1 with different post-stimulation lag periods because the initial velocity of ligand binding is known to correlate with the number of activated receptors (Frojmovic et al., Biophys J, 59, 1991). When control platelets were stimulated with PAR1-AP (50μM), PAC1-binding rates did not change up to 5 min. In P2Y12-deficient platelets, the binding rate in the initial 30 sec was equivalent to control platelets and estimated values of activated αIIbβ3 in the first time fraction was, at least, about 50 % of PMA-treated platelets regardless of P2Y12-signals. In sharp contrast, only a subtle increase of PAC1 binding was seen in the sixth time fraction (2.5–3min), indicating that majority of activated αIIbβ3 was rapidly inactivated within 3 min in P2Y12-deficient platelets. We also found clear, but transient PAC1 binding induced by PAR1 stimulation in megakaryocytic cell line, CMK. When FITC-PAC1 binding was analyzed in CMK expressing high levels of glycoprotein Ib, substantial PAC1 binding was observed 0.5–10 min after PAR1 stimulation. Similar transient PAC1 binding profiles were also observed in primary megakaryocytes derived from cultured umbilical cord blood mononuclear cells. Initial velocities of PAC1 binding with different lag periods confirmed the highest levels of αIIbβ3 activation in the first time fraction (0 to 1 min) both in CMK and in megakaryocytes. Next, we found that P2Y12-dependent signals are defective in CMK because intracellular cyclic AMP levels did not change by ADP in PGE1-treated CMK. Overexpression of P2Y12 by nucleofection, however, resulted in ADP-dependent increase of PAC1 binding by PAR1 stimulation and the extent of ADP-dependent effects correlated with the expression levels of P2Y12 both in CMK and in megakaryocytes. It has been believed that agonist-induced αIIbβ3 activation is absent under P2Y12-defective conditions or in megakaryocytic cell line because bound ligand is not observed in “usual” ligand binding assays. However, analyzing PAC1 binding rates in our experimental systems unravels P2Y12-independent transient αIIbβ3 activation. Furthermore, we demonstrated that the duration of αIIbβ3 activation is prolonged by P2Y12-signals in platelets, CMK and megakaryocytes. Here, this new strategy would let us overcome serious problems existing in “usual” ligand binding assays, providing precise information about dynamic behaviors of integrin αIIbβ3.

Description: Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Description: Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Description: Semaphorin 3A (Sema3A) is a secreted disulfide-bound homodimeric molecule that induces growth cone collapse and repulsion of axon growth in the nervous system. Recently, it has been demonstrated that Sema3A is produced by endothelial cells and inhibits integrin function in an autocrine fashion. In this study, we investigated the effects of Sema3A on platelet function by using 2 distinct human Sema3A chimera proteins. We detected expression of functional Sema3A receptors in platelets and dose-dependent and saturable binding of Sema3A to platelets. Sema3A dose-dependently inhibited activation of integrin αIIbβ3byall agonists examined including adenosine diphosphate (ADP), thrombin, convulxin, phorbol 12-myristate 13-acetate, and A23187. Sema3A inhibited not only platelet aggregation induced by thrombin or collagen but also platelet adhesion and spreading on immobilized fibrinogen. Moreover, Sema3A impaired αIIbβ3-independent spreading on glass coverslips and aggregation-independent granular secretion. Sema3A inhibited agonist-induced elevation of filamentous action (F-actin) contents, phosphorylation of cofilin, and Rac1 activation. In contrast, Sema3A did not affect the levels of cyclic nucleotides or agonist-induced increase of intracellular Ca2+ concentrations. Thus, the extensive inhibition of platelet function by Sema3A appears to be mediated, at least in part, through impairment of agonist-induced Rac1-dependent actin rearrangement.

Description: The platelet integrin IIbβ3 has become a new target for the treatment of pathological thrombosis. It becomes apparent that the affinity of IIbβ3 for its ligands is dynamically regulated by inside-out signaling. However, the components that couple diverse intracellular signals to the cytoplasmic domains of IIbβ3 remain obscure. Employing a chymotrypsin-induced IIbβ3 activation model, we previously proposed the hypothesis that Na+/Ca2 +exchanger (NCX) may be involved in inside-out signaling (Shiraga et al:Blood 88:2594, 1996). In the present study, employing two unrelated Na+/Ca2+ exchange inhibitors, 3′,4′-dichlorobenzamil (DCB) and bepridil, we investigated the role of NCX in platelet activation induced by various agonists in detail. Both inhibitors abolished platelet aggregation induced by all agonists examined via the inhibition of IIbβ3 activation. Moreover, these inhibitors abolished IIbβ3 activation induced by phorbol 12-myristate 13-acetate or A23187. On the other hand, neither of these inhibitors showed apparent inhibitory effects on protein phosphorylation of pleckstrin or myosin light chain, or an increase in intracellular calcium ion concentrations evoked by 0.1 U/mL thrombin. These effects of the NCX inhibitors are in striking contrast to those of protein kinase C inhibitor, Ro31-8220. Biochemical and ultrastructural analyses showed that NCX inhibitors, particularly DCB, made platelets “thrombasthenic”. These findings suggest that the NCX is involved in the common steps of inside-out signaling through integrin IIbβ3.

Description: Platelet IIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and β3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and IIbβ3-mediated intracellular Ca2+ signaling in platelets.

Description: In 1998, Gasbarrini et al reported that in ITP cases with Helicobacter pylori (H.pylori) infection, elevation of platelet counts was observed by eradication of this bacterium. Since then, several reports from Italy and Japan confirmed the elevation of platelet counts after eradication. However, the characteristic background in the H.pylori positive ITP and eradication effects on platelet counts is unclear. On the other hand, reports from Spain, North Europe and USA could not show the evidence that eradication is effective on elevating platelet counts in H.pylori positive ITP. Therefore, we designed a nationwide retrospective study in Japan to evaluate the incidence of H.pylori positive ITP cases and the effects of eradication on platelet counts and to clear above problems. Four hundred and thirty-five ITP cases were enrolled over a period of one and half years (2002. 7~2003.12) from 12 hospitals. H. pylori infection was found in 300 cases(65%), who were significantly (P

Description: Semaphorin (Sema) 3A is a secreted disulfide-bound homodimeric molecule that induces growth cone collapse and repulsion of axon growth in the nervous system. Recently, it has been demonstrated that Sema3A is produced by endothelial cells and it regulates vascular morphogenesis by inhibiting integrin function in an autocrine fashion. Since platelet interaction with endothelial cells is critical for thrombus formation as well as hemostasis, we investigated effects of Sema3A on platelet function. We used two distinct Sema3A fusion proteins in this study: human Sema3A fused to Fc (Sema3A/Fc) and human Sema3A fused to placental alkaline phosphatase (Sema3A/AP). We detected dose-dependent and saturable binding of Sema3A fusion proteins to platelets. Flow cytometric analysis with PAC1 or soluble fibrinogen showed that Sema3A/Fc and Sema3A/AP inhibited activation of αIIbβ3 by all agonists examined, including ADP, thrombin, U46619, convulxin, and PMA. Sema3A also inhibited aggregation induced by thrombin or collagen. Moreover, Sema3A inhibited CD62P and CD63 expression after agonist stimulation, indicating that Sema3A inhibits granular secretion as well as activation of αIIbβ3. However, Sema3A did not inhibited thrombin-induced rapid intracellular calcium increase. Sema3A inhibited platelet adhesion to immobilized fibrinogen partially, and it severely impaired spreading of adhered platelets and even shrinking of spread platelets was observed after addition of Sema3A, suggesting that Sema3A impairs rearrangement of platelet cytoskeleton profoundly. Activation of platelets by thrombin or PAR1 peptide increases F-actin contents, and Sema3A inhibited the agonist-induced elevation of F-actin contents. Sema3A treatment also decreased phosphorylation of cofilin in both resting and thrombin-stimulated platelets, suggesting that Sema3A may keep cofilin in the activated state leading to increase severing of F-actin. Since phosphorylation of cofilin was regulated by LIM-kinase, an effecter of Rac-PAK signaling pathway, we next examined effects of Sema3A on Rac1 activation after thrombin stimulation. PAR1 peptide induced rapid activation of Rac1 in platelets, and Sema3A almost completely inhibited the activation of Rac1. These results suggest that Sema3A inhibits agonist-induced actin rearrangement via Rac1-dependent pathway including phosphorylation of cofilin. In conclusion, we demonstrated that Sema3A binds to platelets and inhibits platelet functions extensively. The inhibition of platelet functions by Sema3A appeared to be mediated at least in part through impairment of agonist-induced Rac1-dependent actin rearrangement.