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Biomedical subjects

Makoto Handa

Publications and source records attributed to Makoto Handa.

28 records · Page 2Linked to original sources
PubMed2003

Bernard-soulier syndrome with a homozygous 13 base pair deletion in the signal peptide-coding region of the platelet glycoprotein Ib(beta) gene.

We report a family with Bernard-Soulier syndrome with a homozygous mutation within the GPIb(beta) gene. The proband was a 24-year-old Japanese male who has suffered from life-long bleeding tendency. The patient's sister also had severe bleeding episodes. The proband and the affected sister had no apparent complications including organic or skeletal anomaly, or mental disturbance. They had thrombocytopenia [(35-40) x 10(9)/l] with giant platelets. In addition to platelet size, electron microscopic analysis revealed abnormalities in the internal structures of platelets. Ristocetin-induced platelet aggregation was defective. Flow cytometric analysis and western blot analysis showed that glycoprotein IX was nearly absent in platelets, whereas GPIb(alpha) and GPV were detectable. Genetic studies revealed a 13 base pair deletion in the signal peptide-coding sequence of GPIb(beta). The deletion would cause a frame-shift, resulting in the appearance of a stop codon following an indifferent polypeptide sequence. Analysis of platelet RNA showed that the mutant GPIb(beta) gene was transcribed. The propositus and his affected sister were homozygous for the deletion, whereas their unaffected father and mother were heterozygotes. The molecular defects of this family would help understand the relevance of GPIb(beta) for complex formation of the glycoprotein Ib/IX/V receptor.

Adult↗
PubMed2003

Basic studies on the clinical applications of platelet-rich plasma.

Platelets, which contain many growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), are being used in clinical applications as platelet-rich plasma (PRP). Only a few studies, however, have been conducted on the growth factors present in PRP and on the clinical applications using the drug delivery system (DDS). For the purpose of clinical application, we first modified the PRP preparation method and assessed the amounts of growth factors contained in the human platelet concentrates. Furthermore, we assessed fibrin glue as a DDS of platelet concentrates. Platelet precipitations were made by twice centrifuging human whole blood. The precipitated platelet was resuspended to yield the platelet concentrates. The growth factor concentrations were measured. Fibrin glue sheets containing this platelet concentrate were implanted in rabbit pinna and samples were obtained for immunostaining (anti-PDGF antibody) to assess the use of PRP over time using the fibrin glue as the DDS. The mean concentration of growth factors present in the platelet concentrates was three times or greater than that of conventional PRP. Furthermore, the results indicated that when the platelet concentrate was used with fibrin glue as a carrier, the contents were released over a period of about 1 week. This raises the possibility that this system may be useful in clinical applications.

Adult↗
PubMed2003

[Measurement of plasma von Willebrand factor cleaving protease in patients with varied thrombotic microangiopathy].

Thrombotic thrombocytopenic purpura(TTP) is a multisystem disorders characterized by thrombocytopenia, microangiopathic hemolytic anemia associated with red cell fragmentation, and neurological and renal symptoms. Plasma of patients with TTP has been shown to contain unusually large von Willebrand factor(vWF) multimers that may cause platelet agglutination in vivo. Recently, a metalloprotease responsible for cleavage of vWF multimers has been isolated from normal human plasma and was found to be deficient in some patients with TTP. We examined the activity of the vWF-cleaving protease(vWF-CP), by modified Furlan's method, in plasma from patients with a familial TTP, 3 acquired TTP, 4 thrombotic microangiopathy(TMA) and 2 veno-occlusive disease(VOD) associated after allo-BMT. Diluted plasma samples of patients were incubated with protease-free vWF purified from normal human plasma, in the presence of urea and barium ions. The extent of vWF degradation was assayed by electrophoresis in SDS-agarose gels and immunoblotting. Activity of vWF-CP from 12 normal plasma have been shown as 77-180%(average 115%), whereas, no vWF-CP(below 5%) was observed in plasma from familial TTP, before and after plasma exchange, although FFP infusion therapy has been effective for this patient to recover thrombocytopenia. In 3 acquired TTP, 2 patients showed lack of vWF-CP activity in plasma, and inhibitors against vWF-CP have been elucidated by plasma cross-mixing test. After extensive plasma exchange and FFP infusion followed by corticosteroid therapy, normal vWF-CP was recovered in plasma from 2 acquired TTP patients. Among BMT patients, plasma from 4 BMT-TMA showed normal vWF-CP activities as 55-111%, whereas plasma from 2 BMT-VOD revealed low vWF-CP activity, as 24% and 37%, respectively. Thus, measurement of vWF-CP is crucial to predict differentiation of primary forms of TMA to establish the pathogenesis in varied endothelial dysfunction.

ADAM Proteins↗
PubMed2002

Shear-induced von Willebrand factor-mediated platelet surface translocation of the CD40 ligand.

BACKGROUND: Platelets, which can adhere to damaged vascular surfaces and release bioactive substances upon activation, may play important roles in regulating local inflammatory responses. We focused on the surface translocation of CD40 ligand (CD40L) molecules when the platelets are exposed to a high shear stress. METHOD: Blood specimens were obtained from eight apparently healthy adult donors. The number of CD40L molecules appearing on the surface of platelets after exposure of platelet-rich plasma to a shear rate of 10,800 s(-1) was determined by quantitative flow cytometry. RESULTS: The number of anti-CD40L IgG molecules bound per platelet increased from 15+/-80/platelet before to 355+/-122/platelet after exposure of the platelets to a shear rate of 10,800 s(-1) (p<0.01), but not after their exposure to the relatively low shear rate of 1200 s(-1). This shear-induced platelet surface translocation of CD40L, mediated by the von Willebrand factor (VWF)-GP Ibalpha interaction, was enhanced in the presence of a low concentration of epinephrine (100 nM), which by itself, however, could not cause platelet activation. Our results demonstrate that fluid force induces the appearance of CD40L on the surface of platelets, and also that this phenomenon is enhanced in the presence of a low concentration of epinephrine, corresponding to that released by sympathetic stimulation.

Adult↗
PubMed2002

Rolling properties of rGPIbalpha-conjugated phospholipid vesicles with different membrane flexibilities on vWf surface under flow conditions.

The recombinant fragment of the platelet membrane glycoprotein, rGPIbalpha, was conjugated to phospholipid vesicles with the average diameter of ca. 1 microm using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). We used five kinds of rGPIbalpha-vesicles with different fluorescent anisotropies of 1,6-diphenyl-1,3,5-hexatriene (DPH) to study the rolling properties of the vesicles on the von Willebrand factor (vWf)-immobilized surface. Under flow conditions, the rolling velocity of the rGPIbalpha-vesicles decreased with the increasing membrane flexibility. It is considered that the vesicles with a high membrane flexibility have a high deformability and can be flattened to a high degree during rolling on the vWf surface, thus resulting in the large contact area. We obtained a recipe to control the rolling velocity of artificial platelets by membrane flexibility.

Blood Platelets↗
PubMed2002

Reconstitution of adhesive properties of human platelets in liposomes carrying both recombinant glycoproteins Ia/IIa and Ib alpha under flow conditions: specific synergy of receptor-ligand interactions.

Liposomes carrying both recombinant glycoprotein Ia/IIa (rGPIa/IIa) and Ib alpha (rGPIb alpha) (rGPIa/IIa-Ib alpha-liposomes) instantaneously and irreversibly adhered to the collagen surface in the presence of soluble von Willebrand factor (VWF) at high shear rates, in marked contrast with translocation of liposomes carrying rGPIb alpha alone on the VWF surface. In the absence of soluble VWF, the adhesion of rGPIa/IIa-Ib alpha-liposomes to the collagen surface decreased with increasing shear rates, similar to liposomes carrying rGPIa/IIa alone. While adhesion of liposomes with exofacial rGPIa/IIa and rGPIb alpha densities of 2.17 x 10(3) and 1.00 x 10(4) molecules per particle, respectively, was efficient at high shear rates, reduction in rGPIb alpha density to 5.27 x 10(3) molecules per particle resulted in decreased adhesion even in the presence of soluble VWF. A 50% reduction in the exofacial rGPIa/IIa density resulted in a marked decrease in the adhesive ability of the liposomes at all shear rates tested. The inhibitory effect of antibody against GPIb alpha (GUR83-35) on liposome adhesion was greater at higher shear rates. Further, the anti-GPIa antibody (Gi9) inhibited liposome adhesion more than GUR83-35 at all shear rates tested. These results suggest that the rGPIa/IIa-collagen interaction dominates the adhesion of rGPIa/IIa-Ib alpha-liposomes to the collagen surface at low shear rates, while the rGPIa/IIa-collagen and rGPIb alpha-VWF interaction complements each other, and they synergistically provide the needed functional integration required for liposome adhesion at high shear rates. This study thus has confirmed for the first time the proposed mechanisms of platelet adhesion to the collagen surface under flow conditions using the liposome system.

Antigens, CD↗
PubMed2002

Carbon monoxide modulates endotoxin-induced microvascular leukocyte adhesion through platelet-dependent mechanisms.

BACKGROUND: Although precise mechanisms remain to be determined, recent studies show that heme oxygenase-1 (HO-1), providing endogenous carbon monoxide (CO) and bilirubin, serves as an antiinflammatory enzyme. This study aimed to clarify roles of CO in regulation of microvascular adhesion of platelets and leukocytes in endotoxemia. METHODS: Rats pretreated with or without hemin were anesthetized with pentobarbital and received continuous infusion of endotoxin. Platelets labeled with carboxyfluorescein diacetate succinimidyl ester and leukocyte behavior in mesenteric venules were visualized using intravital ultra-high-speed intensified fluorescence videomicroscopy. To examine the mechanisms for the effects of HO-1 on platelet and leukocyte behavior during endotoxemia, these studies were repeated with superfusion of either CO, bilirubin, or zinc protoporphyrine-IX. RESULTS: Endotoxin caused a marked depression of platelet velocity traversing along periendothelial regions, accompanied by augmented rolling and adhesion of leukocytes in venules. The endotoxin-elicited changes were attenuated by the HO-1 induction with hemin and restored by blockade of the enzyme activity with zinc protoporphyrine-IX, a potent inhibitor of HO-1. Such an inhibitory action of HO-1 on microvascular cell adhesion was reproduced by local superfusion of the buffer containing CO at micromolar concentrations. Such antiadhesive actions of CO on leukocytes disappeared under immunoneutralization of glycoprotein Ibalpha, an adhesion molecule against platelets, but not against leukocytes. Platelets isolated from hemin-treated rats increased their ability to generate CO and displayed lesser sensitivity of agonist-induced aggregation than those from controls. CONCLUSIONS: These results suggest that CO desensitizes endotoxin-induced adhesive responses of leukocytes, mainly through its ability to ameliorate platelet activation.

Animals↗
PubMed2002

A novel polymorphism, 70Leu/Phe, disrupts a consensus Leu residue within the leucine-rich repeat sequence of platelet glycoprotein Ibalpha.

Platelet glycoprotein (GP) Ib/IX/V complex mediates high-shear dependent platelet activation through an interaction with the von Willebrand factor (vWF). All four subunits of the complex have a structural motif, the leucine-rich repeat (LRR) sequence, with leucines in conserved positions. Here we report a new polymorphism, Leu/Phe at residue 70 of GPIbalpha, which disrupts the consensus sequence of the LRR in the vWF binding domain. Genotype frequencies among 142 healthy Japanese subjects were 92.3%, 7.7%, and 0.0%, for the 70Leu/Leu, 70Leu/Phe, and 75Phe/Phe genotypes, respectively. Ristocetin-induced or shear-induced platelet aggregation was not significantly different between the 70Leu/Leu and 70Leu/Phe genotypes. In in vitro studies, a recombinant GPIbalpha fragment with 70Phe (L70F) as compared to that with 70Leu (WT) had low reactivity to anti-GPIbalpha monoclonal antibodies, GUR20-5 and Hip1, both of which recognize conformation-specific epitopes within the 45-kDa domain. Ristocetin-induced 125I-vWF binding to L70F, however, did not differ from that to WT.

Amino Acid Substitution↗
PubMedDate not supplied

Hemostatic effects of phospholipid vesicles carrying fibrinogen gamma chain dodecapeptide in vitro and in vivo.

We studied prototypes of platelet substitutes that bear on their surface a dodecapeptide, HHLGGAKQAGDV (H12). The peptide is a fibrinogen gamma chain carboxy-terminal sequence (gamma400-411) and recognizes specifically the active form of glycoprotein (GP) IIb/IIIa on the surface of activated platelets. We conjugated H12 to the end of poly(ethylene glycol) chains on the surface of a phospholipid vesicle with an average diameter of 220 nm to prepare H12-PEG-vesicles. The half-life of the H12-PEG-vesicles was significantly prolonged by PEG modification, and the ability of H12 on the surface of the vesicle to recognize GPIIb/IIIa was maintained even though the surface was modified with PEG chains. The H12-PEG-veiscles enhanced the in vitro thrombus formation of platelets that were adhering to a collagen-immobilized plate, when thrombocytopenia-imitation blood was passed over the plate. Based on the flow cytometric analyses of PAC-1 binding and P-selectin expression, the H12-PEG-vesicles were shown not to cause platelet activation. Furthermore, the H12-PEG-vesicles dose-dependently shortened the tail bleeding time of thrombocytopenic rats. It was confirmed that the H12-PEG-vesicles had a hemostatic effect and may be a suitable candidate for an alternative to human platelet concentrates transfused into thrombocytopenic patients.

Animals↗