Morio Arai

Platelets with 10% of the normal amount of glycoprotein VI have an impaired response to collagen that results in a mild bleeding tendency

Platelet glycoprotein VI (GPVI), a 62kD membrane protein, has been identified as one of the platelet receptors for collagen, since GPVI-deficient platelets exhibit abnormal responses to collagen and an abnormal bleeding tendency. We report a female patient with a mild bleeding history whose platelets expressed 10% GPVI of normal platelets. Shape change, aggregation and ATP release of the patients platelets were completely absent in response to 1-5 micrograms/ml collagen but present normally in response to ADP and Ca2+ ionophore A23187. Adhesion of the patients platelets to coated collagen was mildly affected (40-60% of normal platelets) in spite of only 10% expression of GPVI. Flow cytometrical studies revealed that the patients platelets expressed normal amounts of the GPIa/IIa complex. These results suggest that platelet GPVI is less involved in adhesion to collagen than shape change and aggregation induced by collagen.

Involvement of Glycoprotein VI in Platelet Thrombus Formation on Both Collagen and von Willebrand Factor Surfaces Under Flow Conditions

Background—We studied the role of glycoprotein (GP) VI in platelet adhesion and thrombus formation on the immobilized collagen and von Willebrand factor (vWF) surface under flow conditions. Methods and Results—Whole blood obtained from 2 patients with GP VI–deficient platelets and the effects of the Fab of anti–GP VI antibody (Fab/anti–GP VI) were tested. Blood containing platelets rendered fluorescent by mepacrine was perfused on immobilized type I collagen or vWF under controlled wall shear rate. Platelet adhesion and thrombus formation were detected by epifluorescent videomicroscopy. The percentage of surface coverage by the platelets was calculated. Fc receptor &ggr;-chain and spleen tyrosine kinase (Syk) were immunoprecipitated from the lysate of platelets stimulated by vWF plus ristocetin and then analyzed by antiphosphotyrosine immunoblotting. No platelet attachment was seen on the surface of collagen even after 9 minutes of perfusion of blood at relatively low (100 s−1) or high (1500 s−1) wall shear rate, either in the case of blood containing GP VI–deficient platelets or in the presence of Fab/anti–GP VI, whereas significant platelet thrombus formation was noted after control blood perfusion. Such interference with the actions of GP VI also reduced firm platelet adhesion on immobilized vWF. vWF-induced tyrosine phosphorylation of GP VI–associated Fc receptor &ggr;-chain followed by Syk activation occurred in normal platelets, but little activation of Syk occurred in GP VI–deficient platelets. Conclusions—GP VI plays crucial roles in platelet thrombus formation on the surface of collagen under flow conditions in humans and is also involved in the process of firm platelet adhesion on the surface of vWF.

Clinical Trial to Investigate the Pharmacokinetics, Pharmacodynamics, Safety, and Efficacy of Recombinant Factor VIIa in Japanese Patients With Hemophilia With Inhibitors

A multicenter and open-labeled clinical trial of human recombinant factor VIIa (rFVIIa) was conducted in Japanese patients with severe hemophilia A or B with inhibitors. The trial consisted of 2 parts. In study 1, the pharmacokinetics, pharmacodynamics, and safety of a single dose of 120 μg/kg ofrFVIIa were investigated in 8 patients. In the subsequent study 2, the hemostatic effect and safety ofrFVIIa were evaluated during a 24-week period in 10 patients. In study 1, the mean maximum FVII-coagulant activity (FVII:C) was found to occur after 10 minutes; activity then decreased rapidly and returned to the baseline within 24 hours after a single intravenous infusion of rFVIIa. The mean half-life of FVII:C was 3.5 hours. The activated partial thromboplastin time and prothrombin time in the patients were immediately shortened but returned to the baseline within 24 hours after dosing. In study 2, 86 μg/kg to 120 μg/kg ofrFVIIa (mean, 97 μg/kg) was administered 1 to 85 times to 10 patients. A total of 58.0% (91/157) of bleeding episodes were treated excellently or effectively, with 5 (3.2%) ineffective episodes. There was no apparent trend in the relationship of the hemostatic effect with bleeding sites, mean dose, or number of injections. The efficacy rate, however, was significantly higher (90.0%) in bleeding episodes treated within 3 hours than in those treated at longer intervals (31.0%). No treatment-related adverse events were observed, and there was no evidence of antibody formation to rFVIIa. In conclusion,rFVIIa is an effective and well-tolerated option for treatment of bleeding episodes in hemophilia patients with inhibitors.

Substantial expression of glycoproteins IX and V on the platelet surface from a patient with Bernard-Soulier syndrome

Summary. Flow cytometric studies demonstrated that the platelets from a patient with Bernard‐Soulier syndrome expressed substantial amounts of both GPIX and GPV, although in reduced amounts.

Two double heterozygous mutations in the F7 gene show different manifestations

Summary. We sequenced the factor VII gene (F7) in two unrelated Japanese patients with factor VII (FVII) deficiency. In the first (an asymptomatic 46‐year‐old man with FVII activity and antigen levels of 1·2% and 21% of normal respectively), novel E25K and H348Q mutations were identified in the doubly heterozygous state. In transiently transfected HEK293 cells, the level of FVII‐E25K mutant activity in the culture media was significantly lower than that of FVII wild type, whereas the antigen levels of both proteins were similar. This suggests that the E25K mutation is associated with a dysfunctional FVII molecule. In the second patient (a 47‐year‐old woman with FVII activity and antigen levels of less than 1% and 6% respectively), an IVS4+1 mutation and a novel −96C to T transition were detected in the double heterozygous state. In electrophoretic mobility shift assays, the −96T mutation was shown to disrupt binding of Sp1.

Mechanisms of platelet retention in the collagen-coated-bead column.

Although the glass-bead column has been used to measure platelet adhesion, whether platelet interaction with glass beads represents physiologic processes remains unsettled. In an attempt to obtain more physiologic platelet responses, plastic beads coated with type I collagen have been recently developed to replace glass beads. In this study, we analyzed the factors responsible for platelet retention in the collagen-coated-bead column and investigated its possible clinical applications. We pumped citrated whole-blood samples into columns at a fixed speed with an injection pump and calculated platelet-retention rates by measuring platelet counts before and after passage through the columns. The platelet-retention rates, which were highly reproducible with samples from healthy donors, were reduced in a patient with glycoprotein (GP) VI deficiency but not in patients with type III von Willebrand disease. Anti-GPIIb/IIIa antibody and GRGDS peptide markedly inhibited platelet retention, whereas inhibition of the GPIb-von Willebrand factor or GPIa/IIa-collagen interaction had no effect. Data on the effects of various antiplatelet agents (including the antithrombin agent argatroban, prostacyclin, acetylsalicylic acid, and the ADP scavenger creatine phosphate/creatine phosphokinase) support the usefulness of this assay method in clinical application. Our findings suggest that GPVI and GPIIb/IIIa but not the GPIb-von Willebrand factor interaction are mainly involved in platelet retention in this column.

Five novel and four recurrent point mutations in the antithrombin gene causing venous thrombosis.

We analyzed the antithrombin (AT) gene in 9 unrelated Japanese patients with thrombotic disease. All 7 exons, the splice junctions, and the 5′-flanking region of the AT gene were amplified by polymerase chain reaction and sequenced directly. Nine different point mutations, all in the heterozygous state, were identified. Five novel (M-32T, M89K, L146H, Q159X, and L409P) and 2 previously reported (R132X and R359X) point mutations were identified in patients with type 1 deficiency.Two different missense mutations, R393C and R393H, located in the protease reactive site were detected in patients with type 2 deficiency. No other sequence abnormalities in the AT gene were detected by direct sequencing. None of the mutations was present in 100 alleles from 50 unrelated Japanese control subjects. Although type 1 deficiency was diagnosed in patient 7 on the basis of approximately 50% AT antigen and activity levels, the data indicated that the novel L409P mutation is a type 2 pleiotropic effects (PE) deficiency because its location in the C-terminal portion of the reactive site is similar to the locations of reported PE type mutations, and it is highly conserved among other serpins.

Novel Bernard-Soulier syndrome variants caused by compound heterozygous mutations (case I) or a cytoplasmic tail truncation (case II) of GPIbα

A defective platelet glycoprotein (GP) Ib/IX/V complex [von Willebrand factor (VWF) receptor] results in Bernard-Soulier syndrome (BSS), which is characterized by macrothrombocytopenia and impaired ristocetin- and thrombin-induced platelet aggregation. We found 2 independent BSS-variant families: Case I [compound heterozygous mutations, p.Glu331X and a frame shift by a deletion at c.1444delA of GPIbα (GP1BA) terminating at a premature stop codon (p.Thr452ProfsX58)], and case II [homozygous nonsense mutation at c.1723C>T, p.Gln545X]. Case I platelets expressed no GPIbα, resulting in absence of ristocetin-induced platelet aggregation (RIPA) and 50% reduction in thrombin-induced aggregation with no shape change. The mothers platelets had 50% the expression level of A-type GPIbα (4-repeated VNTR: variable number of tandem repeats, p.[Thr145Met; Ser399_Pro411[4]]); the fathers platelets had the same expression level of C-type GPIbα (2-repeated VNTR, p.Ser399_Pro411dup) as the mothers platelets. The mothers RIPA was significantly higher than the fathers. Thrombin-induced aggregation was normal in both parents. Case II platelets expressed a GPIbα with an abnormal cytoplasmic tail, p.Gln545X-truncated GPIbα, which complexed with GPIX and GPV on the cell surface; its expression level of the complex was normal. Case II platelets had reversible RIPA, with no ATP release, and weak thrombin-induced aggregation without shape change. These results suggest that a signaling process through the GPIbα cytoplasmic tail required for full platelet activation is defective in BSS variant case II and a length polymorphism of GPIbα is associated with a modified level of RIPA heterozygous BSS case I.

Successful Management of Massive Intraperitoneal Bleeding in a Hemophilia A Patient with Inhibitor by Surgical Debridement of the Incomplete Hematoma and Administration of Recombinant Factor VIII and Activated Factor VII

hibitor. He had a type 1 (distal) inversion in intron 22 of his FVIII gene. While human immunodeficiency virus and hepatitis B virus antigens were negative, hepatitis C virus RNA was positive in his blood (360 kIU/ml). Liver coagulopathy, however, was not documented. He had had recurrent bleeding episodes including intracranial , gastrointestinal and intraperitoneal bleeding, which were successfully treated with rFVIIa. Despite manual reduction of his inguinal hernia, localized pain persisted. Considering the possibility of incarceration, an inguinal herniorrhaphy was performed as an emergency surgical procedure under rFVIIa cover. Since a hematoma of the major omentum was herniated in the right illiopubic tract, the distal part of the major omentum was resected with a hematoma. An illiopubic tract repair, rather than a tension-free repair using a mesh, was carried out through an open anterior route. Since the patient’s symptom was associated with the major omentum, the incision on the abdominal wall was larger than usual. During the operation, no blood transfusion was necessary, despite moderate difficulties associated with hemostasis, such as oozing at the site of incision or contact, and mild nodular appearance was found on the hepatic surface. rFVIIa (188 g/kg) was administered as an intravenous bolus just prior to the incision, every 2 h until 24 h after the surgery and every 3 h thereafter. Tranexamic acid was infused at 20 mg/kg every 6 h, avoiding concurrent infusion with rFVIIa. Since massive bleeding of more than 1.2 l/day persisted from the abdominal drainage tube postoperatively, resulting in a hypovolemic shock on postoperative day 4, APCC at 50–100 U/kg was additionally administered every 12 h for 4 days mainly to infuse FXa, avoiding concurrent infusion with rFVIIa. The daily schedule was APCC at hours 0 and 12, and rFVIIa at hours 2, 5, 8, 11, 14, 17, 20 and 23. Packed red blood cells, fresh frozen plasma Introduction

Lipid-Protein Interactions

Biological membranes are a fluid mosaic complex composed of various proteins and a phospholipid bilayer, which are the major structural components. Analyses of the interaction between the components of cell membranes and other biological ligands are of importance in understanding the biochemical functions of cell membranes. To aid in addressing these analyses, liposomes (rearranged artificial phospholipid membranes) have been widely utilized. Liposomes are the artificial vesicles in which an aqueous volume solution is enclosed by a membrane composed of phospholipids. Application of liposomes has particular advantages in the analysis of the interaction between the components of the cell membrane and other biological ligands.