Search

Essentials Acquired Glanzmann thrombasthenia (aGT) is generally caused by function-blocking antibodies (Abs). We demonstrated a unique aGT case due to marked reduction of αIIbβ3 with anti-αIIbβ3 Abs. The anti-αIIbβ3 Abs of the patient did not inhibit platelet function but reduced surface αIIbβ3. Internalization of αIIbβ3 induced by the Abs binding may be responsible for the phenotype. SUMMARY: Background Acquired Glanzmann thrombasthenia (aGT) is a bleeding disorder generally caused by function-blocking anti-αIIbβ3 autoantibodies. Aim We characterize an unusual case of aGT caused by marked reduction of surface αIIbβ3 with non-function-blocking anti-αIIbβ3 antibodies (Abs). Methods A 72-year-old male suffering from immune thrombocytopenia since his 50s showed exacerbation of bleeding symptom despite mild thrombocytopenia. Platelet aggregation was absent with all agonists but ristocetin. Analysis of αIIbβ3 expression and genetic analysis were performed. We also analyzed effects of anti-αIIbβ3 Abs of the patient on platelet function and αIIbβ3 expression. Results Surface αIIbβ3 expression was markedly reduced to around 5% of normal, whereas his platelets contained αIIbβ3 to the amount of 40-50% of normal. A substantial amount of fibrinogen was also detected in his platelets. There were no abnormalities in ITGA2B and ITGB3 cDNA. These results indicated that reduced surface αIIbβ3 expression caused a GT phenotype, and active internalization of αIIbβ3 was suggested. Anti-αIIbβ3 IgG Abs were detected in platelet eluate and plasma. These Abs did not inhibit PAC-1 binding, indicating that the Abs were non-function-blocking. Surface αIIbβ3 expression of a megakaryocytic cell line and cultured megakaryocytes tended to be impaired by incubation with the patient's Abs. After 2 years of aGT diagnosis, his bleeding symptom improved and surface αIIbβ3 expression was recovered to 20% of normal with reduction of anti-αIIbβ3 Abs. Conclusion We demonstrated a unique aGT phenotype due to marked reduction of surface αIIbβ3. Internalization induced by anti-αIIbβ3 Abs may be responsible in part for the phenotype.

Background Thrombin belongs to the most potent platelet agonists and activates human platelets through GPIbα and two protease activated receptors (PARs), PAR1 and PAR4. However, the details of thrombin receptor system, especially the role of PAR4 on human platelet activation is still not clear. Objectives We found a significant difference in PAR4-activating peptide (PAR4-AP)-induced, but not PAR1-AP, platelet aggregation between healthy Japanese subjects. Sequencing analysis revealed a single nucleotide change in PAR4 gene F2RL3 (SNP rs773902) leading to Ala120Thr variant. To elucidate the role of PAR4 in human platelet activation, we examined if platelet activation induced by PAR4-AP may be associated with PAR4 genotype. Methods Platelets from 202 healthy Japanese volunteers were genetically analyzed and determined the genotype frequency of rs773902. Agonist induced platelet aggregation, integrin αIIbβ3 activation, granule release, Ca2+ mobilization, and activation of ERK and MLC were evaluated. The specificity of effects observed in platelets was confirmed in 293T cells transfected PAR4-Thr120 or Ala120. Results The frequencies of PAR4 variant Thr/Thr120, Ala/Thr120, and Ala/Ala 120 were 5.9, 37.1, and 57.0%, respectively. Platelets with Thr/Thr120 showed significantly higher reactivity in PAR4-AP-induced platelet aggregation, αIIbβ3 activation and granule release compared to platelets with Ala/Ala120. PAR4-AP induced higher Ca2+ mobilization and ERK activation in platelets with Thr/Thr120 than Ala/Ala120. Ca2+ mobilization and ERK activation were also increased in 293T cells transfected with PAR4-Thr120 compared to Ala120. Conclusion Our data suggested that PAR4-AP-induced platelet reactivity between PAR4 rs773902 was associated with altered intensity of Ca2+ mobilization and ERK activation.

Affinity regulation of integrin αIIbβ3 for fibrinogen by inside-out signaling plays a critical role in hemostasis. Calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) was identified as a Rap1-activating molecule, and its role in inside-out αIIbβ3 activation was established in CalDAG-GEFI-deficient mice. However, little information regarding CalDAG-GEFI in human platelets is available. Here, we report a 16-year-old girl with CalDAG-GEFI deficiency who has been suffering from severe bleeding tendency. Although talin and kindlin-3 were normally detected, CalDAG-GEFI was undetectable in her platelets by western blotting. Genetic analysis revealed compound heterozygous CalDAG-GEFI mutations, Lys309X and Leu360del, which were responsible for CalDAG-GEFI deficiency. The functional analysis demonstrated impaired αIIbβ3 activation by various agonists except for phorbol myristate acetate, normal calcium mobilization, and impaired Rap1 activation, which were consistent with the phenotype of CalDAG-GEFI-deficient mice. Despite substantial αIIbβ3 activation at high agonist concentrations, she had severe bleeding tendency. Further functional analysis demonstrated markedly delayed αIIbβ3 activation velocity and decreased shear-induced thrombus formation. Contrary to CalDAG-GEFI-deficient mice, which showed integrin-dependent neutrophil functional abnormality, neutrophil β2 integrin activation was not impaired in the patient. Our results demonstrate the critical role of CalDAG-GEFI in rapid αIIbβ3 activation of human platelets.

Thrombin belongs to the most potent platelet agonists and activates human platelets through GPIbα and two protease activated receptors (PARs), PAR1 and PAR4. However, the details of thrombin receptor system, especially the role of PAR4 on human platelet activation is still not clear.We found a significant difference in PAR4-activating peptide (PAR4-AP)-induced, but not PAR1-AP, platelet aggregation between healthy Japanese subjects. Sequencing analysis revealed a single nucleotide change in PAR4 gene F2RL3 (SNP rs773902) leading to Ala120Thr variant. To elucidate the role of PAR4 in human platelet activation, we examined if platelet activation induced by PAR4-AP may be associated with PAR4 genotype.Platelets from 202 healthy Japanese volunteers were genetically analyzed and determined the genotype frequency of rs773902. Agonist induced platelet aggregation, integrin αIIbβ3 activation, granule release, CaThe frequencies of PAR4 variant Thr/Thr120, Ala/Thr120, and Ala/Ala 120 were 5.9, 37.1, and 57.0%, respectively. Platelets with Thr/Thr120 showed significantly higher reactivity in PAR4-AP-induced platelet aggregation, αIIbβ3 activation and granule release compared to platelets with Ala/Ala120. PAR4-AP induced higher CaOur data suggested that PAR4-AP-induced platelet reactivity between PAR4 rs773902 was associated with altered intensity of Ca

Background: Kindlin-3 which is expressed mainly in hematopoietic cells, is essential for platelet fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) activation and kindlin-3 deficiency causes severe bleeding problems. αIIbβ3 activation is tightly regulated by inside-out signaling, and the direct interaction of talin and kindlin-3 with β3-cytoplasmic tail following CalDAG-GEFI-induced Rap1 activation is critical for αIIbβ3 activation. We have reported that immediate and sustained αIIbβ3 activation by inside-out signaling is important for thrombus formation under physiological conditions (Blood 2016). However, the details of inside-out signaling are not still fully understood. Recently we identified patient with kindlin-3 deficiency as the first Japanese patient with kindlin-3 deficiency caused by novel p.W277X nonsense mutation. To clarify the role of kindlin-3 and the molecular mechanism of inside-out signaling, we analyzed single platelet behavior adhered on collagen under physiological flow conditions, and established kindlin-3 deficient human erythroleukemia HEL cell line. Case: The patient was 8-month old female, born to Japanese consanguineous parents. She has been suffering from bleeding tendency shortly after birth. Her peripheral blood showed slightly decreased platelet count (95x103/L), anemia (Hb 6.9 g/L), and elevated leukocyte count of 37.2x106/L with 1.0% blast. Although the surface expressions of glycoproteins were comparable to healthy control, her platelet aggregations and αIIbβ3 activation were strongly impaired in all agonist stimulations due to defective kindlin-3 expression. The sequencing analysis revealed the homozygous novel nonsense mutation, p.W277X (c.918G>A) in kindlin-3. Methods: Human platelets were obtained from healthy control subjects (CT) and patients with kindlin-3 deficiency and Glanzmann thrombasthenia (GT). Whole blood was perfused at a shear rate 1,250s-1 on collagen surface and shear-induced in vitro thrombus formation during 10 minutes was observed. To evaluate the single platelet behavior after the initial attachment on collagen, each single platelet was analyzed by CellTracker software (Pccinini F. et al. Bioinformatics 2015). To further determine the role of kindlin-3 in inside-out signaling, Kindlin-3 was knocked out by CRISPR-Cas9 system and established kindlin-3 deficient HEL cell line. Results: First, we compared shear-induced thrombus formation between CT, GT, and kindlin-3 deficiency. In contrast to the stable and large platelet aggregate formation in CT after 10 minutes blood perfusion, almost no aggregate was observed in both GT and Kindlin-3 deficiency. In kindlin-3 deficiency, the initial platelet attachment on collagen seemed comparable to that of CT and GT. However, the single adherent platelets looked unstable. Next, we determined the behavior of initially attached platelets. Between CT, GT, and kindlin-3 deficiency, the numbers of platelets attached on collagen during 10 seconds were comparable. Between the initially attached platelets, 31.25% of CT platelets formed stable adhesion followed by platelet aggregation. Similar to CT, 33% of GT platelets adhered stably. However, these GT platelets did not proceed to aggregate formation due to αIIbβ3 deficiency. In contrast to GT, kindlin-3 deficient platelets showed increased detachment, only 9% of initially attached platelets stably adhered. These results suggest that kindlin-3 is indispensable for platelet initial adhesion to collagen by integrin α2β1 activation and explains severe bleeding symptoms in kindlin-3 deficiency than GT. To further investigate how kindlin-3 contributes in initial platelet adhesion to collagen, we established kindlin-3 deficient HEL cell line. In contrast to parental HEL cells, PMA stimulation did not induce αIIbβ3 activation in kindlin-3 deficient HEL cells suggesting impaired inside-out signaling. The introduction of wild type kindlin-3 cDNA, but not W227X mutant, rescued αIIbβ3 activation. Conclusion: The detailed analyses by tracking single adherent platelet on collagen confirmed the role of kindlin-3 in initial step of physiological thrombus formation. Newly established kindlin-3 deficient HEL cell line is useful for further exploration of the role of kindlin-3 and inside-out signaling in platelets and expected contribution for development of new antiplatelet therapy. Disclosures Tomiyama: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria; Kyowa-Kirin: Honoraria.

Many different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbβ3, whereas inside-out signaling (αIIbβ3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbβ3. We evaluated the time-dependent changes of the αIIbβ3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbβ3 activation, whereas PAR4-activating peptide induced long-lasting αIIbβ3 activation. When αIIbβ3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbβ3. Compared with control platelets, the dissociation of α-actinin from αIIbβ3 was only transient in PAR4-stimulated P2Y12-deficient platelets in which the sustained αIIbβ3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbβ3 and inhibited PAR1-induced initial αIIbβ3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbβ3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbβ3 activation by inside-out signaling.

Introduction: Integrin αIIbβ3 plays an essential role in platelet aggregation, and quantitative or qualitative defects in αIIbβ3 lead to severe bleeding diathesis, which is known as Glanzmann thrombasthenia (GT). Although platelet counts and morphology are usually normal in GT patients, recent several reports demonstrate that constitutive gain-of-function mutations around transmembrane regions of αIIb or β3 are associated with congenital macrothrombocytopenia, which is referred to as αIIbβ3-related thrombocytopenia. αIIb(R995W) is the most prevalent mutation in αIIbβ3-related thrombocytopenia among Japanese (Kunishima, Kashiwagi, et al. Blood. 2011;117:5479-84). We also found reduction of surface expression of αIIbβ3 on platelets in patients with αIIbβ3-rerated thrombocytopenia, suggesting that activating mutations may affect platelet function via not only activating state of αIIbβ3 but also its expression (Kashiwagi, et al. Mol Genet Genomic Med. 2013;1:77-86). However, the pathogenesis of thrombocytopenia caused by αIIbβ3 gain-of-function mutations is not fully understood. We have generated αIIb(R990W), which is corresponding to human αIIb(R995W), knock-in (KI) mice and previously reported that αIIb(R990W) KI mice showed mild macrothrombocytopenia and Homo KI mice revealed marked reduction of surface αIIbβ3 expression in platelet and impaired platelet function like GT (ASH meeting 2014). In this study, we further analyzed αIIb(R990W) KI mice to elucidate the mechanism causing macrothrombocytopenia. Methods: αIIb(R990W) mutation was introduced to C57BL/6-derived ES cells by homologous recombination and KI mice were generated. Reticulated platelets were detected by flow cytometry after thiazole orange staining. Serum thrombopoietin (TPO) levels were measured by ELISA. Platelet life span was assessed by flow cytometry after injection with NHS-biotin. Platelet counts and reticulated platelets were monitored after injection of murine TPO or anti-GPIb antibody. We counted megakaryocytes (MgK) in tissue preparation of femur and spleen. MgK ploidy was analyzed by flow cytometry. We analyzed proplatelet formation (PPF) of cultured fetal liver cells from embryonic day 14.5 embryos. Results: Platelet counts were decreased in KI mice [WT: 1.22 ± 0.11 (x106/μL), Hetero: 1.10 ± 0.14*, Homo: 0.91 ± 0.11* (*p Conclusion: Our results suggest that the αIIbβ3 gain-of-function mutation, αIIb(R990W), leads to thrombocytopenia via mainly impaired thrombopoiesis. There was no difference in number and ploidy of MgK. However, Homo KI mice show marked reduction of PPF, indicating impaired proplatelet formation appears to be a main cause of impairment of thrombopoiesis by the mutation. Disclosures: No relevant conflicts of interest to declare. Disclosures Kanakura: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Tomiyama:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Consultancy.

Platelet integrin alphaIIbbeta3 activation is tightly controlled by intracellular signaling pathways, and several molecules, including talin, have been identified as critical for alphaIIbbeta3 activation. However, the whole pathway associated with alphaIIbbeta3 activation remains to be determined. To address this issue, we established a Chinese hamster ovary cell line (parental cells) that expresses constitutively activated chimeric integrin alphaIIbalpha6Bbeta3, and then obtained mutant cells expressing inactivated alphaIIbalpha6Bbeta3 by genome-wide mutagenesis. We have performed expression cloning to isolate signaling molecules responsible for integrin activation in the mutant cells. We show that integrin-linked kinase (ILK) complements defective integrin activation in the mutant cells. ILK mRNAs in the mutant cells contained 2 nonsense mutations, R317X and W383X, in a compound heterozygous state, resulting in a complete loss of ILK expression. Moreover, the mutant cells showed partially impaired activation of endogenous beta1 integrins. Knockdown of ILK in parental cells significantly suppressed the activated state of alphaIIbalpha6Bbeta3. However, ILK overexpression did not rescue the impaired integrin activation in talin knocked-down parental cells, whereas overexpression of talin-F3, a subdomain of the talin head domain, restored the function. Our present data suggest that ILK contributes to inside-out integrin activation.

Introduction Genetic deficiencies of PROS1, PROC, and SERPINC1 (antithrombin) are risk factors for deep vein thrombosis (DVT). Diagnosis of the inherited deficiencies of these three genes is sometimes difficult because of the phenotypic variability. This study was undertaken to reveal the frequency of nonsynonymous mutations of these three genes in Japanese DVT patients. Patients/Methods One hundred seventy-three DVT patients were registered by the Sub-group of Blood Coagulation Abnormality, from the Study Group of Research on Measures for Intractable Diseases. We sequenced the entire coding regions of the three genes in all DNA samples and identified the nonsynonymous mutations. Results and Conclusions For PROS1 we identified 15 nonsynonymous mutations in 28 DVT patients; for PROC, 10 nonsynonymous mutations in 17 patients; and for SERPINC1, 13 nonsynonymous mutations in 14 patients. Five patients had two mutations in PROS1 and PROC, and all of them had PROS1 K196E mutation. We previously identified one patient with a large PROS1 gene deletion. Thus, 55 out of 173 patients (32%) carried at least one genetic defect in the three genes. The PROS1 K196E mutation found in 15 Japanese DVT patients was the most prevalent. Mutations of PROC K193del and V339M were the second, each found in four patients. Our data suggested that the PROC K193del mutation caused the loss of the anticoagulant activity but not the amidolytic activity. Our effort is the first DNA resequencing study to identify the genetic variations in DVT patients without any consideration of their plasma activities and antigens. To minimize selection bias in a future evaluation of the contribution of genetic deficiency to DVT, we must recruit patients consecutively.

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protease inhibitor that inhibits the tissue factor-initiated blood coagulation cascade reactions. Based on these anticoagulant functions of TFPI, we hypothesized that genetic variations in TFPI may alter the TFPI expression or impair the anticoagulant function and could predispose persons to deep vein thrombosis (DVT). This study was undertaken to examine whether the genetic polymorphisms in TFPI are associated with the plasma TFPI levels and risk for DVT. We sequenced the entire coding regions of TFPI in 175 Japanese DVT patients and identified 12 genetic variants, including one missense mutation, Asn221Ser. The missense mutation occurred at the site presumably attached to the glycosylphosphatidylinositol anchor in the TFPI-beta form. The allele frequency of the mutant Ser-coding allele of the Asn221Ser mutation was 8% in the Japanese general population consisting of 1684 individuals. The Asn221Ser mutation was significantly associated with the total TFPI levels (Asn/Asn, n = 108, total TFPI = 56.57 +/- 0.88 ng/ml (mean +/- SD) vs. Asn/Ser + Ser/Ser, n = 16, total TFPI = 63.44 +/- 2.28 ng/ml, P = 0.0058). The genotype was not associated with the free TFPI level. This Asn221Ser mutation was not associated with DVT. Thus, the Asn221Ser mutation occurring in the TFPI-beta form was associated with the total TFPI level, but not a risk for DVT. The absence of the putative glycosylphosphatidylinositol anchor in TFPI-beta under pathological conditions remains to be studied.

The dose required for the anticoagulant effect of warfarin exhibits large inter-individual variations. This study sought to determine the contribution of four genes, vitamin K epoxide reductase ( VKORC1 ), γ-glutamyl carboxylase ( GGCX ), calumenin ( CALU ), and cytochrome P450 2C9 ( CYP2C9 ) to the warfarin maintenance dose required in Japanese patients following ischemic stroke. We recruited 93 patients on stable anticoagulation with a target International Normalized Ratio (INR) of 1.6–2.6. We genotyped eleven representative single nucleotide polymorphisms (SNPs) in the three genes involved in vitamin K cycle and the 42613A>C SNP in CYP2C9 , known as CYP2C9 ⁎ 3 , and then examined an association of these genotypes with warfarin maintenance doses (mean ± SD = 2.96 ± 1.06 mg/day). We found an association of effective warfarin dose with the − 1639G>A ( p = 0.004) and 3730G>A genotypes ( p = 0.006) in VKORC1 , the 8016G>A genotype in GGCX ( p = 0.022), and the 42613A>C genotype in CYP2C9 ( p = 0.015). The model using the multiple regression analysis including age, sex, weight, and three genetic polymorphisms accounted for 33.3% of total variations in warfarin dose. The contribution to inter-individual variation in warfarin dose was 5.9% for VKORC1 − 1639G>A, 5.2% for CYP2C9 42613A>C, and 4.6% for GGCX 8016G>A. In addition to polymorphisms in VKORC1 and CYP2C9 , we identified GGCX 8016G>A, resulting in the missense mutation R325Q, as a genetic determinant of warfarin maintenance dose in Japanese patients.

The congenital form of thrombotic thrombocytopenic purpura (TTP) is caused by genetic mutations in ADAMTS13. Some, but not all, congenital TTP patients manifest renal insufficiency in addition to microangiopathic hemolysis and thrombocytopenia. We included 32 congenital TTP patients in the present study, which was designed to assess whether congenital TTP patients with renal insufficiency have predisposing mutations in complement regulatory genes, as found in many patients with atypical hemolytic uremic syndrome (aHUS). In 13 patients with severe renal insufficiency, six candidate complement or complement regulatory genes were sequenced and 11 missense mutations were identified. One of these missense mutations, C3:p.K155Q mutation, is a rare mutation located in the macroglobulin-like 2 domain of C3, where other mutations predisposing for aHUS cluster. Several of the common missense mutations identified in our study have been reported to increase disease-risk for aHUS, but were not more common in patients with as compared to those without renal insufficiency. Taken together, our results show that the majority of the congenital TTP patients with renal insufficiency studied do not carry rare genetic mutations in complement or complement regulatory genes.

ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation through cleavage of von Willebrand factor (VWF) multimers. In humans, genetic or acquired deficiency in ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP), a condition characterized by thrombocytopenia and hemolytic anemia with microvascular platelet thrombi. In this study, we report characterization of mice bearing a targeted disruption of the Adamts13 gene. ADAMTS13-deficient mice were born in the expected mendelian distribution; homozygous mice were viable and fertile. Hematologic and histologic analyses failed to detect any evidence of thrombocytopenia, hemolytic anemia, or microvascular thrombosis. However, unusually large VWF multimers were observed in plasma of homozygotes. Thrombus formation on immobilized collagen under flow was significantly elevated in homozygotes in comparison with wild-type mice. Thrombocytopenia was more severely induced in homozygotes than in wild-type mice after intravenous injection of a mixture of collagen and epinephrine. Thus, a complete lack of ADAMTS13 in mice was a prothrombotic state, but it alone was not sufficient to cause TTP-like symptoms. The phenotypic differences of ADAMTS13 deficiencies between humans and mice may reflect differences in hemostatic system functioning in these species. Alternatively, factors in addition to ADAMTS13 deficiency may be necessary for development of TTP.

Objective— Obesity is a common risk factor in insulin resistance and cardiovascular diseases. Although hypoadiponectinemia is associated with obesity-related metabolic and vascular diseases, the role of adiponectin in thrombosis remains elusive. Methods and Results— We investigated platelet thrombus formation in adiponectin knockout (APN-KO) male mice (8 to 12 weeks old) fed on a normal diet. There was no significant difference in platelet counts or coagulation parameters between wild-type (WT) and APN-KO mice. However, APN-KO mice showed an accelerated thrombus formation on carotid arterial injury with a He-Ne laser (total thrombus volume: 13.36±4.25×10 7 arbitrary units for APN-KO and 6.74±2.87×10 7 arbitrary units for WT; n=10; P −1 , as well as platelet aggregation induced by low concentrations of agonists, was enhanced in APN-KO mice, and recombinant adiponectin inhibited the enhanced platelet aggregation. In WT mice, adenovirus-mediated overexpression of adiponectin additionally attenuated thrombus formation. Conclusion— Adiponectin deficiency leads to enhanced thrombus formation and platelet aggregation. The present study reveals a new role of adiponectin as an endogenous antithrombotic factor.

Summary. Background: The αIIbβ3 antagonists inhibit platelet aggregation and are used as antithrombotic agents for cardiothrombotic disease. The present study investigates the correlation of inhibition of fibrinogen and von Willebrand factor (VWF) binding by αIIbβ3 antagonists with the inhibition of platelet aggregation and prolongation of bleeding time (BT). Methods: Inhibition of fibrinogen and VWF binding were assessed in a purified αIIbβ3-binding assay. As an in vitro cell-based assay, platelet aggregation and VWF-mediated adhesion studies were performed using human platelets. In vivo effects on BT were measured using a template device in dogs at the same time as an ex vivo aggregation study was performed. Results: In vitro studies demonstrated that the antiaggregatory effects of αIIbβ3 antagonists correlate with their inhibition of fibrinogen binding, but not VWF. Interestingly, the effects of αIIbβ3 antagonists on BT could be differentiated from the inhibition of platelet aggregation. Furthermore, this differentiation was strongly correlated with the different inhibitory potencies between fibrinogen and VWF binding, as well as that between VWF-mediated adhesion and aggregation. Conclusions: Our study provides novel evidence showing that the inhibitory effect of αIIbβ3 antagonists on VWF, but not fibrinogen binding, correlates with their ability to prolong BT.

CD47 (integrin-associated protein) serves as a receptor for thrombospondin-1 (TSP-1) and Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), and the TSP-1/CD47 interaction has been believed to augment integrin-mediated platelet function. Here, employing SHPS-1-immunoglobulin (Ig) as a ligand, we have newly demonstrated that CD47 acts as an inhibitory receptor for platelet function. The binding of SHPS-1-Ig was solely mediated by CD47, because CD47-deficient platelets failed to bind murine SHPS-1-Ig. The human SHPS-1/CD47 interaction inhibited the platelet aggregation induced by several kinds of agonists at a low concentration. Moreover, human SHPS-1 expressed on the cell surface as well as soluble SHPS-1-Ig markedly inhibited the platelet spreading on, but not initial adhesion to, immobilized fibrinogen. Again, neither murine SHPS-1 expressed on the cell surface nor murine SHPS-1-Ig inhibited the spreading of CD47-deficient platelets. We further investigated the tyrosine phosphorylation of signaling proteins during platelet spreading on immobilized fibrinogen. Unexpectedly, SHPS-1 inhibited alpha(IIb)beta(3)-mediated platelet spreading without disturbing focal adhesion kinase (FAK) tyrosine phosphorylation. Further examination revealed that SHPS-1 inhibited the tyrosine phosphorylation of alpha-actinin, a downstream effector of FAK, but not of cortactin. Thus, it is likely that the SHPS-1/CD47 interaction inhibits alpha(IIb)beta(3)-mediated outside-in signaling by interfering with the downstream pathway of FAK. Taken together, our data suggest that SHPS-1 negatively regulates platelet function via CD47, especially alpha(IIb)beta(3)-mediated outside-in signaling.

SummaryThe crystal structure of αvβ3 in complex with a cyclic RGDcontaining ligand has recently been demonstrated. However, the functional significance of each residue within ligand binding loops has not been fully elucidated. Here, by employing alaninescanning mutagenesis, we have examined the functional role of ligand contact residues in αv. Tyr178 –> Ala substitution (Tyr178Ala) and Asp218Ala abolished a monovalent ligand, WOW-1 Fab binding as well as soluble fibrinogen binding, which is in perfect agreement with the crystallography. However, Asp150Ala showed no or only a modest inhibition of ligand binding. In contrast, Tyr substitution at Ala215 (Ala215Tyr) increased WOW-1 Fab binding, suggesting that the substitution increased the integrin affinity. The adhesion assay to immobilized fibrinogen showed essentially the same data as obtained using soluble ligands. Our present data indicate that Tyr178 and Asp218, but not Asp150 in αv is critically involved in ligand-binding and that Ala215 could regulate the affinity of αvβ3.

Affinity/avidity state of integrin alpha IIb beta 3 is regulated by intracellular inside-out signaling. Although several megakaryocytic cell lines have been established, soluble ligand binding to alpha IIb beta 3 expressed in these cells by cellular agonists has not been demonstrated. We have re-examined agonist-induced alpha IIb beta 3 activation on megakaryocytic cell lines with a marker of the late stage of megakaryocytic differentiation, glycoprotein Ib (GPIb). Activation of alpha IIb beta 3 was assessed by PAC1 and soluble fibrinogen binding to the cells. We found that alpha IIb beta 3 expressed in CMK cells with high GPIb expression was activated by a phorbor ester, phorbol myristate acetate (PMA). Although the population of the GPIbhigh cells was0.5% of the total cells, incubation with a nucleoside analog, ribavirin, efficiently increased the PMA-reactive GPIbhigh cells. Not only PMA but also a calcium ionophore, A23187, induced alpha IIb beta 3 activation, and PMA and A23187 had an additive effect on alpha IIb beta 3 activation. Ligand binding to the activated alpha IIb beta 3 in the GPIbhigh CMK cells is totally abolished by an alpha IIb beta 3-specific antagonist, and inhibited by wortmannin, cytochalasin-D and prostaglandin E1, and the effects of these inhibitors on alpha IIb beta 3 activation in the GPIbhigh CMK cells were compatible with those in platelets. We have also demonstrated that the ribavirin-treated CMK cells express PKC-alpha, -beta, -delta and -theta, and suggested that PKC-alpha and/or -beta appear to be responsible for PMA-induced activation of alpha IIb beta 3 in CMK cells.

αIIbβ3 and αvβ3 belong to the β3integrin subfamily. Although the β3 subunit is a key regulator for the biosynthesis of β3 integrins, it remains obscure whether missense mutations in β3 may induce the same defects in both αIIbβ3 and αvβ3. In this study, it is revealed that thrombasthenic platelets with a His280Pro mutation in β3, which is prevalent in Japanese patients with Glanzmann thrombasthenia, did contain significant amounts of αvβ3 (about 50% of control) using sensitive enzyme-linked immunosorbent assay. Expression studies showed that the His280Proβ3 mutation impaired αIIbβ3 expression but not αvβ3 expression in 293 cells. To extend these findings, the effects of several β3 missense mutations leading to an impaired αIIbβ3expression on αvβ3 function as well as expression was examined: Leu117Trp, Ser162Leu, Arg216Gln, Cys374Tyr, and a newly created Arg216Gln/Leu292Ser mutation. Leu117Trp and Cys374Tyr β3 mutations did impair αvβ3 expression, while Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Ser mutations did not. With regard to ligand binding function, Ser162Leu mutation induced especially distinct effects between 2 β3 integrins: it markedly impaired ligand binding to αIIbβ3 but not to αvβ3 at all. These data clearly demonstrate that the biosynthesis and the ligand binding function of αIIbβ3 and those of αvβ3 are regulated in part by different mechanisms. Present data would be a clue to elucidate the regulatory mechanism of expression and function of β3 integrins.

Localization of epitopes for platelet-associated (PA) anti–GPIIb-IIIa (αIIbβ3) autoantibodies in chronic immune thrombocytopenic purpura remains elusive. Previous studies suggest that PA antibodies recognize the tertiary structure of intact glycoprotein (GP) IIb-IIIa. To localize their epitopes using antigen-capture enzyme-linked immunosorbent assay (ELISA), the reactivity of 34 PA anti–GPIIb-IIIa antibodies was examined with recombinant GPIIb-IIIa having a defect in ligand-binding sites in either GPIIb or GPIIIa, and no major conformational change was induced: KO variant GPIIb-IIIa was attributed to a 2–amino acid insertion between residues 160 and 161 in the W3 4-1 loop in GPIIb, and CAM variant GPIIb-IIIa was attributed to D119Y in GPIIIa. In one third (11 of 34) of the patients, PA antibodies showed a marked decrease (less than 50%) in reactivity with KO compared with wild-type GPIIb-IIIa. Their reactivity was also impaired against GPIIbD163A-IIIa. In sharp contrast, they reacted normally with CAM GPIIb-IIIa. OP-G2, a ligand-mimetic monoclonal antibody, markedly inhibited their binding to GPIIb-IIIa in patients with impaired binding to KO GPIIb-IIIa, but small GPIIb-IIIa antagonists did not. In addition, a newly developed sensitive ELISA indicated that autoantibodies showing impaired binding to KO are more potent inhibitors for fibrinogen binding. The present data suggest that certain PA anti–GPIIb-IIIa autoantibodies recognize epitopes close to the ligand-binding site in GPIIb, but not in GPIIIa.

In chronic immune thrombocytopenic purpura (ITP), anti-GPIIb-IIIa (alphaIIbbeta3) autoantibodies have been detected in serum and/or platelet-associated IgG (PAIgG) and considered as one of the major causes. We examined whether anti-alphavbeta3 antibodies might be present in ITP cases because of the similarity between alphavbeta3 and GPIIb-IIIa (alphaIIbbeta3). Modified antigen capture ELISA (MACE) using human umbilical vein endothelial cells (HUVEC) showed the presence of serum anti-alphavbeta3 antibodies in 23 of 80 ITP patients (29%). Cross-adsorption studies between platelets and HUVEC demonstrated that most of anti-alphavbeta3 and anti-GPIIb-IIIa antibodies exclusively reacted with alphavbeta3 and GPIIb-IIIa, respectively. Platelet-associated anti-GPIIb-IIIa antibodies did not react with alphavbeta3, either. Interestingly, patients having anti-alphavbeta3 antibodies showed significantly lower platelet counts than negative patients. These results indicate the serum anti-alphavbeta3 antibodies are different ones from the classical anti-GPIIb-IIIa (alphaIIbbeta3) antibodies and would provide a new insight into the pathophysiology of ITP as well as the autoantigenic epitopes on beta3 integrins.

Integrin αvβ3 has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in αv, a non–I-domain α subunit, remain to be identified. Recently in αIIb, the other partner of the β3 subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in 293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of αv in ligand binding. Wild-type αvβ3 was able to bind soluble fibrinogen following integrin activation either by 0.5 mM manganese dichloride (MnCl2) or a mutation of β3 threonine (T)562 to asparagine. However, mutation of tyrosine (Y)178 to alanine in the predicted G172-G181 loop of αv abolished fibrinogen binding, and alanine (A) substitutions at adjacent residues phenylalanine (F)177 and tryptophan (W)179 had a similar effect. Cells expressing Y178Aαvalso failed to bind to immobilized fibrinogen. Moreover, the Y178A mutation abolished the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-αvβ3 antibody, and the expression of β3 ligand–induced binding sites (LIBS) induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp contrast to the data obtained with αIIb, none of the mutations in the predicted W3 4-1 loop in αv impaired ligand binding. These results implicate αv Y178 in ligand binding to αvβ3, and they suggest that there are key structural differences in the adhesive ligand-binding sites of αvβ3 and αIIbβ3.

SummaryBernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. This complex is composed of four subunits, GPIbα, GPIbβ, GPIX and GPV. We describe here the genetic basis of the disorder in a patient with BSS. Flow cytometric analysis of the patient’s platelets showed greatly reduced GPIbα and GPIX surface expression. Immunoblot analysis disclosed absence of GPIbα, GPIbβ and GPIX in the platelets. DNA sequencing analysis revealed a novel missense mutation in the GPIbβ gene that converts Pro (CCG) to Arg (CGG) at residue 74. Homozygosity of the mutation was confirmed by allele-specific restriction analysis, chromosome 22 microsatellite analysis and quantitative Southern blotting. The mutant GPIbβ was normally transcribed. Transient transfection studies confirmed that mutant GPIbβ impairs surface expression of GPIb/IX, showing that the mutation is responsible for a BSS phenotype observed in the patient.

Bernard-Soulier syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. We describe here the genetic basis of the disorder in a patient with BSS. Flow cytometric analysis of the patient's platelets showed a greatly reduced GPIbα and completely absent GPIX surface expression. Immunoblot analysis disclosed greatly reduced GPIbα and residual amounts of GPIbβ and GPIX in the platelets. DNA sequencing analysis revealed the patient to be homozygous for a novel missense mutation in the GPIX gene that converts Cys (TGT) to Tyr (TAT) at residue 97. Transient transfection studies confirmed that mutant GPIX was not expressed on the transfected cells, showing that the mutation was responsible for the BSS phenotype observed in the patient.

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 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 alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; 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 beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), 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 beta3 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.

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.

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.

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.

This study investigated the difference of [Ca 2+ ] i movement in platelets in response to thrombin and TRAP. The involvement of a IIb b 3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca 2+ ] i . The second peak of [Ca 2+ ] i , but not the first peak was abolished by the inhibition of platelet aggregation with a IIb b 3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA 2 receptor antagonist, BM13505, also abolished the second peak of [Ca 2+ ] i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB 2 demonstrated that a IIb b 3 antagonists and aspirin almost completely inhibited the production of TXB 2 . In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca 2+ ] i even in the presence of platelet aggregation, and a high level of [Ca 2+ ] i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with a IIb b 3 antagonists had no effect on [Ca 2+ ] i change and TXB 2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca 2+ ] i response and TXA 2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to a IIb b 3 are responsible for the synthesis of TXA 2 which results in the induction of the second peak of [Ca 2+ ] i in low thrombin- but not TRAP-stimulated platelets.

Integrin subunit compatibility within and between species plays a major role in heterodimer assembly and ligand specificity. As an example, human alphaIIb pairs only with human beta3 and does not assemble a heterodimer with beta3 from other species. We use interspecies subunit chimeras to identify molecular requirements for subunit compatibility and show that species-restricted heterodimer assembly depends on a unique hexapeptide VGSDNH in an extended loop of the hypothetical human beta3 MIDAS domain. This allows us to express alphaIIb(1-233) and beta3(111-318) as a soluble, mini-integrin that retains RGD-dependent ligand recognition. Thus, in the case of one integrin, alphaIIbbeta3, the molecular requirements for integrin subunit compatibility and ligand recognition are intimately related.

In the present study, we have investigated the mechanism of affinity modulation of alpha IIb beta 3 by chymotrypsin. We first confirmed that alpha-chymotrypsin could activate alpha IIb beta 3 (approximately 7,000 molecules per platelet) without major intracellular signaling. However, we unexpectedly found that high concentrations of amiloride dose-dependently inhibited 125I-fibrinogen binding to the chymotrypsin-treated platelets, as well as the platelet aggregation (IC50 [50% inhibitory concentration] for fibrinogen binding, 530 mumol/L). In contrast, amiloride did not inhibit alpha IIb beta 3 activation induced by anti-alpha IIb beta 3 monoclonal antibody PT25–2 or AP5. To identify the pathway involved, the effects of alteration of Na+ gradient in platelets were examined. Lowering Na+ gradient by replacing extracellular Na+ with tetramethylammonium (TMA) increased the number of activated alpha IIb beta 3 by twofold, as assessed by fibrinogen-binding assay. The incubation of platelets with ouabain, a Na+/K(+)- adenosine triphosphatase (ATPase) inhibitor, further augmented alpha IIb beta 3 activation. These data suggested that a likely candidate for the pathway was Na+/Ca2+ exchanger. At 140 mmol/L [Na+]o, 45Ca2+ influx to the chymotrypsin-treated platelets was twofold greater than that to non-treated platelets. Replacement of Na+ with TMA further increased the Ca2+ influx, and the increase was inhibited by amiloride dose-dependently. 3′,4′-Dichlorobenzamil (DCB) and bepridil, relatively specific inhibitors of Na+/Ca2+ exchanger, also inhibited the chymotrypsin-induced alpha IIb beta 3 activation, and the IC50 values of these inhibitors for fibrinogen binding were 25 mumol/L and 52 mumol/L, respectively. Moreover, platelet aggregation induced by various physiologic agonists was inhibited by DCB or bepridil, while platelet agglutination by ristocetin was not. Our data newly suggest that Na+/Ca2+ exchanger operating in reverse mode may be directly involved in inside-out signaling that activates alpha IIb beta 3.

Background & objectives: Thrombin is the most important physiological agonist for platelet thrombus formation. In human platelets, two thrombin receptors, protease activated receptor 1 (PAR1) and PAR4, elicit intraplatelet signals through G-protein for platelet activation. Although PAR1 has been established as a major thrombin receptor, the role of PAR4 in human platelet activation is so far not clear. Genetically modified mice are critical tool for human platelet research. However, the different thrombin receptor system in murine platelets which have PAR3 and PAR4 prevents applying the information of murine platelets for human platelets. To explore the role of PAR4, we investigated inter-individual variation of platelet activation induced by PAR4 and PAR4 dependent signaling in Japanese subjects. Methods: Human platelets obtained from healthy volunteers were stimulated by PAR1-activating peptide (PAR1-AP) and PAR4-AP, and detailed aggregation responses were analyzed by aggregometer. Platelet integrin αIIbβ3 activation, granule release and calcium mobilization were measured by flow cytometry. Platelet signaling induced by PAR4-AP was analyzed by Western blotting. Sequencing analysis of PAR4 was performed using RNA and genomic DNA isolated from peripheral mononuclear cells. PAR4 expression vector was transfected to 293T cells, and calcium mobilization was analyzed by flow cytometry. Results: Sequencing analysis between control subjects revealed heterogeneity of PAR4 polymorphism c.533G>A (p.Ala120Thr). The further sequencing analysis of PAR4 in 143 Japanese revealed that the frequencies of 120A/A, A/T, and T/T were 53.8, 40.6, and 5.6% respectively. Subjects who have PAR4-120T required significantly lower concentrations of PAR4-AP to induce irreversible aggregation compared to subjects with PAR4-120A (n=24, p Conclusions: We demonstrate the significant difference in PAR4-AP induced platelet aggregation, αIIbβ3 activation, granule release, calcium mobilization, and ERK phosphorylation between platelets with PAR4-120A and -120T. Our results suggest the association of PAR4 polymorphism with human platelet reactivity in response to PAR4-AP in Japanese. Disclosures Kanakura: Eisai: Research Funding; Alexionpharma: Research Funding; Chugai Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Shionogi: Research Funding; Fujimotoseiyaku: Research Funding; Toyama Chemical: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding.

Within the native integrin alpha pi b beta 3, the conformation of the amino-terminal domain of the beta 3 subunit has a significant influence on the availability of the Leu33/Pro33 polymorphism that defines the A1 and A2 alleles, respectively, of the P1A alloantigen system. The majority of anti-P1A1 IgG antibodies, affinity-purified by adsorption to either (A1/A1)-platelets or purified alpha pi b(A1) beta 3, fail to bind to the Leu33 polymorphic loop within Cys26-Cys38 in native beta 3 unless this sequence is maintained in a proper orientation by two noncontiguous human beta 3 sequences. By comparing IgG binding to recombinant beta 3 molecules composed of segments of human, Xenopus and avian sequences expressed by Spodoptera frugiperda cell lines, we have found that neither region alone is sufficient for full expression of the epitope. One sequence, that most proximal to Leu33/Pro33, lies within beta 3[54-133], and a second distal sequence is located within beta 3[435-490]. Proximity of the distal sequence to the Cys26-Cys38 loop in native beta 3 is confirmed by the fact that both anti-P1A1 IgG and the murine monoclonal antibody SZ21, specific for beta 3[28-35], completely inhibit the binding of rabbit polyclonal IgG specific for the sequence beta 3[479-485]. Anti-P1A1 IgG antibodies from all of the 7 donors selectively bind to the epitope within the native conformation of beta 3. However, in one of the seven donors, roughly 60% of the anti-P1A1IgG binds exclusively to denatured (A1) beta 3, and not to either denatured (A2) beta 3 or nondenatured (native) (A1) beta 3. This IgG subpopulation cannot be purified by adsorption to intact (A1/A1)-platelets or to native alpha pi b(A1) beta 3 integrin. The physiologic relevance of this exceptional type of anti-P1A1 antibody is debatable, but the presence of IgG with this specificity only in an immunized subject suggests that the epitope may well be presented on a "denatured" form of beta 3 in vivo. Regardless, our results indicate that the amino-terminal domain of the native beta 3 molecule associates with noncontiguous beta 3 sequences, and the epitope recognized by a majority of anti-P1A1 antibodies is a sensitive indicator of correct tertiary structure within this region of beta 3.

We have compared the binding of affinity-purified anti-PlA1 IgG from seven nonrelated donors with chimeric integrin subunit beta 3 molecules expressed in the baculovirus-Spodoptera frugiperda insect cell system. Beta 3 chimeras were engineered to include segments of antigenic human beta 3 sequences spliced to intervening segments of nonantigenic Xenopus beta 3 sequence. Our results clearly show that antibodies from all seven donors will bind to nondenatured molecules containing the antigenic human beta 3 Cys26-Cys38 loop only when it is presented in a correct orientation that must be maintained by noncontiguous human sequences. Key downstream sequences are located within the region beta 3(288–490), flanking either side of the putative long-range disulfide at Cys435. Although our results confirm unambiguously that the Leu/Pro polymorphism at position 33 in human beta 3 is necessary for the expression of PlA epitopes, they also indicate that this polymorphic sequence alone is not sufficient. The requirement for additional human beta 3 sequence transcends the need to maintain a correct orientation within the Cys26-Cys38 loop itself, because the murine monoclonal antibody SZ21, which recognizes the sequence beta 3(28–35) contained within the Cys26-Cys38 loop, binds to all chimeras containing this loop, even if the same chimeras are not recognized by anti-PlA1. Our results indicate that additional noncontiguous residues encompassed by the sequence 288–490 either directly contribute to the composition of the PlA1 epitope or, more likely, maintain the Cys26-Cys38 loop in a proper orientation with respect to the remainder of the beta 3 molecule and thereby maintain proper antigenic presentation of the sequences in that loop.

Changes in ligand binding ability of the integrin alpha IIb beta 3 can be monitored by the concomitant expression of ligand-inducible binding sites (LIBS). A new LIBS, the hexapeptide sequence GPNICT (residues 1-6) at the amino terminus of beta 3 recognized by the murine monoclonal antibody (mAb) AP5, is sensitive both to the binding of ligand and to micromolar differences in divalent cation levels. Calcium or magnesium can completely inhibit the binding of AP5 to alpha IIb beta 3 on platelets, with ID50 values of 80 and 1500 microM, respectively. The inhibitory effect of calcium plus magnesium is cumulative. In the presence of 1 mM calcium plus 1 mM magnesium, the peptide RGDW overcomes this inhibition and induces maximal binding of AP5. Maximal AP5 binding is also induced by a molar excess of EDTA. The unique location of the AP5 LIBS was determined by comparing the binding of LIBS-specific mAb to recombinant human-Xenopus beta 3 chimeras produced in a baculovirus expression system. AP5 defines one region at the amino terminus beta 3 1-6. A second region, defined by mAb D3GP3, is probably located within beta 3 422-490, confirming the finding of Kouns et al. (Kouns, W. C., Newman, P.J., Puckett, K. J., Miller, A. A., Wall, C. D., Fox, C. F., Seyer, J. M., and Jennings, L. K. (1991) Blood 78, 3215-3223). The third region, encompassing at most residues 490-690, and perhaps more precisely located within 602-690 (Du X., Gu, M., Weise, J. W., Nagaswami, C., Bennett, J. S., Bowditch, R., and Ginsberg, M. H. (1993) J. Biol. Chem. 268, 23087-23092), is recognized by the four mAb, anti-LIBS2, anti-LIBS3, anti-LIBS6, and P41. Since its exposure is uniquely regulated by both divalent cations and ligand, the amino terminus of beta 3 may be involved in control of ligand binding by divalent cation mobilization.

CD36 deficiency is divided into two subgroups: neither platelets nor monocytes express CD36 (type I deficiency), and monocytes express CD36 in spite of the lack of platelet CD36 (type II deficiency). We have already demonstrated that a 478C-->T substitution (proline90-->serine) in platelet CD36 cDNA predominates in type II deficiency (Kashiwagi, H., S. Honda, Y. Tomiyama, H. Mizutani, H. Take, Y. Honda, S. Kosugi, Y. Kanayama, Y. Kurata, and Y. Matsuzawa. 1993. Thromb. Haemostasis. 69:481-484). In this study, we revealed that monocyte CD36 cDNA from two type II deficient subjects was heterozygous for C478 and T478 form, while platelet CD36 cDNA of these subjects consisted of only T478 form. In a type I deficient subject, both platelet and monocyte CD36 cDNA showed only T478 form. Expression assay using C478 or T478 form of CD36 cDNA transfected cells revealed that there was an 81-kD precursor form of CD36, and that the maturation of the 81-kD precursor form to the 88-kD mature form of CD36 was markedly impaired by the substitution. The mutated precursor form of CD36 was subsequently degraded in the cytoplasm. These results indicate that the 478C-->T substitution directly leads to CD36 deficiency via defects in posttranslational modification, and that this substitution is the major defects underlying CD36 deficiency.

Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Approximately 10% of cases are classified as atypical due to the absence of Shiga toxin-producing bacteria as a trigger. Uncontrolled activation of the complement system plays a role in the pathogenesis of atypical HUS (aHUS). Although many genetic studies on aHUS have been published in recent years, only limited data has been gathered in Asian countries. We analyzed the genetic variants of 6 candidate genes and the gene deletion in complement factor H (CFH) and CFH-related genes, examined the prevalence of CFH autoantibodies and evaluated the genotype-phenotype relationship in 10 Japanese patients with aHUS. We identified 7 causative or potentially causative mutations in CFH (p.R1215Q), C3 (p.R425C, p.S562L, and p.I1157T), membrane cofactor protein (p.Y189D and p.A359V) and thrombomodulin (p.T500M) in 8 out of 10 patients. All 7 of the mutations were heterozygous and four of them were novel. Two patients carried CFH p.R1215Q and 3 other patients carried C3 p.I1157T. One patient had 2 causative mutations in different genes. One patient was a compound heterozygote of the 2 MCP mutations. The patients carrying mutations in CFH or C3 had a high frequency of relapse and a worse prognosis. One patient had CFH autoantibodies. The present study identified the cause of aHUS in 9 out of 10 Japanese patients. Since the phenotype-genotype correlation of aHUS has clinical significance in predicting renal recovery and transplant outcome, a comprehensively accurate assessment of molecular variation would be necessary for the proper management of aHUS patients in Japan.

Platelet integrin α IIb β 3 activation is regulated by inside-out signaling via agonist stimulation. However, when α IIb β 3 was exogenously expressed in cell lines such as Chinese hamster ovarian cells, physiological agonists hardly induced α IIb β 3 activation. To overcome this disadvantage, we characterized the functional regulation of endogenously expressed α IIb β 3 in a megakaryoblastic cell line, CMK, employing an initial velocity assay for PAC-1 binding. We firstly demonstrated that protease-activated receptor 1-activating peptide induced robust, but transient α IIb β 3 activation in CMK cells with high glycoprotein-Ib expression. Stable talin-1 or kindlin-3 knockdown cells confirmed that the protease-activated receptor 1-activating peptide–induced α IIb β 3 activation was dependent on talin-1 and kindlin-3 expression. In sharp contrast to exogenously expressed α IIb β 3 in Chinese hamster ovarian cells, transient overexpression of full-length talin (FL-talin) or talin-head domain (THD) alone did not activate α IIb β 3 in CMK cells, but required agonist stimulation. Similarly, kindlin-3 overexpression alone did not induce α IIb β 3 activation, but it significantly augmented agonist-induced α IIb β 3 activation. Several mutants of FL-talin and THD suggested that the head–rod interaction was critical for autoinhibition of talin-1, and the interaction between the THD and the membrane-proximal region of the β 3 cytoplasmic tail was essential for talin-mediated α IIb β 3 activation. In addition, THD and kindlin-3 cooperatively augmented protease-activated receptor 1–induced α IIb β 3 activation. We proposed that the CMK cell line is an attractive platform for investigating agonist-, talin-1-, and kindlin-3- dependent α IIb β 3 activation.

Platelet-associated (PA) IgG autoantibodies play an essential role in primary immune thrombocytopenia (ITP). However, little is known about the epitopes of these Abs. This study aimed to identify critical binding regions for PA anti-αIIbβ3 Abs. Because PA anti-αIIbβ3 Abs bound poorly to mouse αIIbβ3, we created human-mouse chimera constructs. We first examined 76 platelet eluates obtained from patients with primary ITP. Of these, 26 harbored PA anti-αIIbβ3 Abs (34%). Further analysis of 15 patients who provided sufficient materials showed that the epitopes of these Abs were mainly localized in the N-terminal half of the β-propeller domain in αIIb (L1-W235). We could identify 3 main recognition sites in the region; 2 eluates recognized a conformation formed by the W1:1-2 and W2:3-4 loops, 5 recognized W1:2-3, and 4 recognized W3:4-1. The remaining 4 eluates could not be defined by the binding sites. Within these regions, we identified residues critical for binding, including S29 and R32 in W1:1-2; G44 and P45 in W1:2-3; and P135, E136, and R139 in W2:3-4. Of 11 eluates whose recognition sites were identified, 5 clearly showed restricted κ/λ-chain usage. These results suggested that PA anti-αIIbβ3 Abs in primary ITP tended to recognize highly restricted regions of αIIb with clonality.

Summary. Background: Thromboxane A2 receptor (TXA2R) abnormality appears to dominantly disturb platelet function. Objectives: To reveal a molecular genetic defect in a patient with TXA2R abnormality and investigate the mechanism for the impaired response to TXA2. Patient: The proband (OSP-2, PT) was a 7-year-old Japanese girl, suffering from repeated mucocutaneous bleeding. Methods and results: U46619 (2.5 and 10 μm)-induced platelet aggregation was remarkably impaired in the proband and her father. Immunoblots showed that TXA2R expression levels in their platelets were approximately 50% of controls, and nucleotide sequence analysis revealed that they were heterozygous for a novel mutation, c.167dupG in the TXA2R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA2R. We then examined αIIbβ3 activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained αIIbβ3 and Rap1B activation in the proband. In addition, platelet secretion as monitored by P-selectin expression was markedly impaired in response to U46619 but not to ADP. The interaction between secreted ADP and P2Y12 has been shown to play a critical role in the sustained αIIbβ3 activation (Kamae et al. J Thromb Haemost 2006; 4: 1379). As expected, small amounts of exogenous ADP (0.5 μm) partially restored the sustained αIIbβ3 activation induced by U46619. Conclusion: Our present data strongly suggest that the impaired platelet activation in response to U46619 in the heterozygous subject for the TXA2R mutation is, at least in part, as a result of the decrease in ADP secretion.

Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown. In this study, we examined whether vinculin would activate integrin in Chinese hamster ovary (CHO) cells expressing human integrin αIIbβ3. Silencing of vinculin by lentiviral transduction with a short hairpin RNA sequence affected the binding of PAC-1 (an antibody recognizing activated human αIIbβ3) to a constitutively active form of αIIbβ3 (α6Bβ3) expressed on CHO cells, while its inhibitory effects were much weaker than those of talin-1. Overexpression of an active form of vinculin without intramolecular interactions, but not the full length one, induced PAC-1 binding to native αIIbβ3 expressed on CHO cells in a manner dependent on talin-1. On the other hand, silencing of talin-1, but not vinculin, failed to induce cell spreading of α6Bβ3-CHO cells on fibrinogen, even in the presence of PT 25-2, a monoclonal antibody that activates αIIbβ3. Thus, an active form of vinculin could induce αIIbβ3 inside-out signaling through the actions of talin-1, while vinculin was dispensable for outside-in signaling.

To determine the mechanism by which platelet counts increase after corticosteroid therapy for human immune thrombocytopenic purpura (ITP), we studied the platelet kinetics using prednisolone (PDN)-treated ITP- prone mice, (NZW x BXSB) F1 (W/B F1). An increase in platelet counts was observed in W/B F1 mice (n = 10, mean +/- SD, 1,202 +/- 202 x 10(3)/microL) 4 weeks after treatment with PDN (2 mg/kg/d); no increase occurred in nontreated W/B F1 mice (n = 5,651 +/- 126, P less than .005). Prolonged platelet life-spans (PLSs) were observed in treated W/B F1 mice (1.29 +/- 0.40 days), but not in nontreated controls (0.60 +/- 0.24 days, P less than .01). No increase in platelet production (platelet turnover) was found in PDN-treated W/B F1 mice, but significant decreases in platelet-associated antibodies (PAAs) and platelet-bindable serum antibodies (PBAs) were noted. Studies on organ localization of radiolabeled platelets showed that hepatic uptake significantly decreased in the treated W/B F1 mice, but not in nontreated W/B F1 mice. To elucidate the effect of PDN on the reticulo- endothelial phagocytic activity in W/B F1 mice, we studied in vivo clearance of IgG-sensitized, 51Cr-labeled autologous erythrocytes. W/B F1 mice treated with PDN showed a marked impairment of their ability to clear these cells, although PDN had little effect on the number of splenic or hepatic macrophage Fc gamma receptors. These results and our previous findings of splenectomy suggest that PDN improves platelet counts not only by suppressing systemic reticulo-endothelial phagocytic function but also by reducing antibody production.

By two-colour flow cytometric analysis, we examined the proportion of B lymphocytes bearing CD5 cell surface antigen (CD 5+ B cells), which are capable of producing autoantibodies, both in peripheral blood and spleen from patients with chronic immune thrombocytopenic purpura (ITP). The percentage of CD5+ B cells in peripheral blood lymphocytes (PBLs) was significantly increased (P less than 0.005) in patients with ITP (3.7 +/- 2.2%, n = 30) as compared with normal controls (1.7 +/- 0.7%, n = 28). However, there was no correlation between the percentages of circulating CD5+ B cells and platelet counts. The percentage of splenic CD5+ B cells in ITP patients was much more increased (9.0 +/- 4.5%, n = 9), P less than 0.005) compared with that of other disorders (3.2 +/- 0.5%, n = 5). Furthermore, isolated splenic CD5+ B cells from two out of five ITP patients produced high levels of IgM-type, platelet-bindable antibodies (PBIgM) after stimulation with Staphylococcus aureus Cowan I (SAC), while CD5- B cells isolated from the same spleen or splenic CD5+ B cells from other non-autoimmune disorders failed to produce significant amount of PBIgM. In three ITP patients, no increase in PBIgM was detected despite SAC stimulation. The increased proportion of CD5+ B cells in peripheral blood and spleen, and their ability to produce anti-platelet antibodies indicate that they are directly involved in the autoimmune pathogenesis in ITP.

We have already demonstrated that Naka antigen (GPIV) is expressed on the monocytes in platelet-Naka- subjects. To define the molecular basis for the discrepancies of GPIV expression between monocytes and platelets in Naka- subjects, we studied whether there are differences in monocyte GPIV mRNA obtained from Naka- and Naka+ subjects, and compared these monocyte GPIV mRNA with Naka- platelet GPIV mRNA which we have recently reported.In Northern blot analysis, monocyte GPIV mRNA from two platelet-Naka- subjects exhibited the same size of 2.9kb and nearly the same amounts as that from platelet-Naka+ subject. And restriction enzyme analysis of GPIV cDNAs which were amplified by a reverse transcription polymerase chain reaction (RT-PCR) showed predictable fragment length.We have already shown that RT-PCR analysis of GPIV mRNA from both Naka- and Naka+ platelets and restriction enzyme analysis of these PCR products show no apparent differences between them. Thus, we conclude that GPIV mRNA from both monocytes and platelets in Naka- subjects has no apparent abnormalities and seems to be the same as that in Naka+ subjects.

Summary Two human hybridomas secreting antiplatelet autoantibodies were established by somatic cell fusion using splenocytes from two patients with chronic idiopathic thrombocytopenic purpura (ITP). These monoclonal antibodies, HT7F and HT8C, were of the IgM isotype and reacted with autologous and allogeneic platelets fixed with paraformaldehyde (PFA). They also bound to fresh platelets. An elution study showed that eluates from allogeneic platelets reacted with autologous platelets. These results indicated that HT7F and HT8C were autoantibodies recognizing a site on the platelet surface. Both monoclonal antibodies were able to induce complement activation in vitro. HT7F was demonstrated to bind to a platelet protein having a molecular mass of 105 kDa under both nonreducing and reducing conditions. No human hybridoma synthesizing antibody against 105 kDa platelet protein has been reported to date. These antibodies may play a role in the pathogenesis of thrombocytopenia in some ITP patients.

Summary Platelet antigens that bind platelet-associated autoantibodies in chronic idiopathic thrombocytopenic purpura (ITP) were demonstrated using a direct immunoprecipitation procedure. ITP platelets, with bound autoantibodies, were radiolabelled and solubilized, and then platelet antigen-antibody complexes adsorbed to protein A-bearing Staphylococcus aureus were analysed by 7.5% sodium dodecyl sulphate, polyacrylamide gel electrophoresis (SDS-PAGE). Direct immunoprecipitation demonstrated the presence of platelet-associated autoantibodies against glycoprotein (GP) IIb/IIIa in four of six ITP patients with an intensive band corresponding to platelet-associated IgG. These results were confirmed by indirect immunoprecipitation using ether eluates from two ITP patients. In addition, only direct immunoprecipitation demonstrated the presence of autoantibodies against an unidentified protein having a molecular mass of 56 kDa in three of the six patients. These three ITP patients having autoantibodies against GP IIb/IIIa and against the 56 kDa protein were studied after splenectomy. Two patients, showing disappearance of autoantibodies against these antigens, attained a complete remission, and one patient, with autoantibodies against the 56 kDa protein despite splenectomy, attained only partial remission. These data suggest that autoantibodies against GP IIb/IIIa and against the 56 kDa protein may play a role in platelet destruction in some ITP patients.