Tadashi Kamiya

Congenital Deficiency of α2-Plasmin Inhibitor Associated with Severe Hemorrhagic Tendency *

alpha(2)-Plasmin inhibitor (alpha(2)PI) is a recently characterized, fast-reacting plasmin inhibitor in human plasma that appears to play an important role in regulation of in vivo fibrinolysis. We report here a case of complete deficiency of alpha(2)PI in man. The patient, a 25-yr-old Japanese man, had a life-long severe bleeding tendency (hemarthrosis and excessive bleeding after trauma). The following tests were within normal limits: platelet count, bleeding time, thrombin time, prothrombin time, partial thromboplastin time, titers of known clotting factors, platelet glass bead retention, Factor VIII-related antigen, platelet aggregation by ADP, collagen and ristocetin, and clot retraction. Routine liver function tests were also normal. The only abnormal finding was that whole blood clot lysis was extemely rapid and was complete in 4-8 h. The concentration of plasma protease inhibitors, including alpha(2)-macro-globulin, antithrombin III, alpha(1)-antitrypsin, and C1INH, were all normal. The concentration of alpha(2)-PI in the patients plasma, assayed by immunological methods, was <0.1 mg/100 ml (normal concentration, 6.1+/-0.88 mg/100 ml [mean+/-SE]) and functional assays showed a complete deficiency of alpha(2)PI. Addition of purified alpha(2)PI to the patients whole blood completely corrected the accelerated fibrinolysis. The patients parents, four siblings, and four other members of this family were asymptomatic, but the titers of alpha(2)PI in their plasmas were congruent with50% of normal pooled plasma. There were three consanguineous marriages in this family, and the alpha(2)PI deficiency appears to have been inherited as an autosomal recessive trait. We speculate that alpha(2)PI deficiency in this patient has led to uninhibited in vivo fibrinolysis that probably causes the severe hemorrhagic tendency. Thus, this study indicates the important role of alpha(2)PI in hemostasis.

Immunofluorescence analysis of neutrophil nonmuscle myosin heavy chain-A in MYH9 disorders: association of subcellular localization with MYH9 mutations.

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly, Sebastian syndrome, and Fechtner syndrome, are rare human disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like cytoplasmic inclusions in granulocytes. Epstein syndrome is another autosomal dominant macrothrombocytopenia associated with Alport syndrome but without leukocyte inclusions. These disorders are caused by mutations in the same gene, the MYH9, which encodes the nonmuscle myosin heavy chain-A (NMMHCA). The term, MYH9 disorders, has been proposed, but the clinicopathologic basis of MYH9 mutations has been poorly investigated. In this study, a total of 24 cases with MYH9 disorders and suspected cases were subjected to immunofluorescence analysis by a polyclonal antibody against human platelet NMMHCA. Abnormal subcellular localization of NMMHCA was observed in every neutrophil from individuals with MYH9 mutations. Comparison with May-Grünwald-Giemsa staining revealed that the NMMHCA always coexisted with the neutrophil inclusion bodies, suggesting that NMMHCA is associated with such bodies. In three cases, neutrophil inclusions were not detected on conventional May-Grünwald-Giemsa-stained blood smears but immunofluorescence analysis revealed the abnormal NMMHCA localization. In contrast, cases with Epstein syndrome and the isolated macrothrombocytopenia with normal NMMHCA localization had no MYH9 mutations. An antibody that recognizes the C-terminal 12 mer peptides showed similar immunoreactivity from the patients heterozygous for truncated mutations that abolished the C-terminal epitope, suggesting that normal NMMHCA dimerizes with abnormal NMMHCA to form inclusion bodies. We further propose that the localization pattern can be classified into three groups according to the number, size, and shape of the fluorescence-labeled NMMHCA granule. Immunofluorescence analysis of neutrophil NMMHCA is useful as a screening test for the clear hematopathologic classification of MYH9 disorders.

Identification of six novel MYH9 mutations and genotype-phenotype relationships in autosomal dominant macrothrombocytopenia with leukocyte inclusions

AbstractThe autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly (MHA), Sebastian syndrome (SBS), and Fechtner syndrome (FTNS), are rare platelet disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like leukocyte inclusions. The locus for these disorders was previously mapped on chromosome 22q12.3–q13.2 and the disease gene was recently identified as MYH9, the gene encoding the nonmuscle myosin heavy chain-A. To elucidate the spectrum of MYH9 mutations responsible for the disorders and to investigate genotype–phenotype correlation, we examined MYH9 mutations in an additional 11 families and 3 sporadic patients with the disorders from Japan, Korea, and China. All 14 patients had heterozygous MYH9 mutations, including three known mutations and six novel mutations (three missense and three deletion mutations). Two cases had Alport manifestations including deafness, nephritis, and cataracts and had R1165C and E1841K mutations, respectively. However, taken together with three previous reports, including ours, the data do not show clear phenotype–genotype relationships. Thus, MHA, SBS, and FTNS appear to represent a class of allelic disorders with variable phenotypic diversity.

Genetic abnormalities of Bernard-Soulier syndrome.

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. BSS is characterized by giant platelets, thrombocytopenia, and prolonged bleeding time, and the hallmark of this disorder is the absence of ristocetininduced platelet agglutination. In the last 10 years, the molecular and genetic bases of many GPIb/IX/V defects have been elucidated, providing a better understanding of primary hemostasis and structure-function relations of the complex. Thus far, more than 30 mutations of the GPIbα, GPIbβ, or GPIX genes have been described in BSS. Recent studies also have shown that the phenotypes caused by mutations in the subunits of the GPIb/IX/V span a wide spectrum, from the normal phenotype, to isolated giant platelet disorders/macrothrombocytopenia, to full-blown BSS and platelet-type von Willebrand disease. Although recent progress in molecular biology has clarified the genotype-phenotype relationships of the GPIb/IX/V disorders, a close examination of platelet morphology on blood smears is still indispensable for a proper diagnosis. In this review, we summarize recent advances in the molecular basis of BSS with special emphasis on giant platelets and the genetic characteristics of Japanese BSS.Int J Hematol. 2002; 76: 319-327.

α2-PLASMIN-INHIBITOR DEFICIENCY (MIYASATO DISEASE)

A 25-year-old man, born in Okinawa, Japan, had a haemorrhagic diathesis characterised by prolonged bleeding and ecchymoses after minor trauma and spontaneous joint haemorrhage. The frequency and severity of these episodes were reduced by an antiplasminic drug. Routine coagulation studies revealed no abnormalities except for significantly sshortened euglobulin-lysis time and whole-blood clot lysis time. Activities of all known clotting and fibrinolytic factors were within normal ranges but no circulating alpha2-plasmin inhibitor was found in the plasma. alpha2-plasmin inhibitor is a potent and fast-acting protease inhibitor. Studies of family members indicated that this abnormality was inherited as an autosomal and recessive gene.

Platelet Function in Bilateral Acute Retinal Necrosis

We tested platelet function in seven patients (four men and three women, ranging in age from 26 to 57 years) with bilateral acute retinal necrosis. Hyperaggregation was detected in six of the seven. Antiplatelet therapy (aspirin, 500 mg/day) together with corticosteroids or other drugs produced satisfactory results. Despite administration of antiplatelet drugs, two retinal detachments developed; both were surgically repaired. A lower dosage of antiplatelet agents in one case and late administration of it in another were thought to have caused the retinal detachments. After the acute stage was over or the retina was reattached, there were no further recurrences during a follow-up period of more than one year.

Mapping of a gene for May-Hegglin anomaly to chromosome 22q.

Abstract. May-Hegglin anomaly (MHA) is a rare autosomal dominant platelet disorder characterized by the triad of giant platelets, thrombocytopenia and leukocyte inclusions. Both the molecular and the genetic defects responsible for this disorder remain unknown. In order to map the gene responsible for MHA, we performed a genome-wide linkage study using highly polymorphic short tandem repeat markers in a single Japanese MHA family. Significant linkage was obtained for the markers on the long arm of chromosome 22 (22q12.3–q13.2), with a maximum two-point lod score of 4.52 at a recombination fraction of 0.00 for the markers D22S1142 and D22S277. Haplotype analysis mapped a critical region for the disease locus to a 13.6-centimorgan region, between D22S280 and D22S272. The relative proximity of the platelet GPIbβ gene (22q11.2) to this region, as well as its involvement in an isolated giant platelet disorder, suggested a possible involvement of GPIbβ mutations in MHA. However, DNA-sequencing analysis in two patients revealed no abnormality in the sequence of the GPIbβ gene. This is the first report of linkage for MHA, and further analysis of this locus may lead to the identification of a gene the product of which regulates platelet and leukocyte morphology.

Adsorption of Anaphylatoxins and Platelet‐Specific Proteins by Filtration of Platelet Concentrates with a Polyester Leukocyte Reduction Filter

Anaphylatoxins generated during storage of platelet concentrates (PCs) may potentially have side effects on platelet transfusion. We evaluated the anaphylatoxin‐scavenging abilities of white blood cell reduction filters. Among the commercially available filters for PCs, one made with polyester fiber (PL50) dramatically adsorbed C3a and C4a anaphylatoxins to the respective mean level of 1,721–208 ng/ml and 1,240–141 ng/ml in 3‐day‐old PCs. C3a and C4a were measured as the native and des Arg form of each complement by radioimmunoassay. C3a and C4a anaphylatoxins in the supernatant plasma fraction from 3‐day‐old PC again decreased from 1,136 to 114 ng/ml and from 1,086 to 65 ng/ml, respectively. The filter also adsorbed 85% of platelet factor 4 (PF4) and 31% of β‐thromboglobulin (β‐TG), which had been released from platelets into the plasma during storage. The plasma levels of adhesive proteins such as fibronectin, fibrinogen, and von Willebrand factor, and plasma lactate dehydrogenase activity did not decrease after filtration. Another polyester filter (PL5A), on the other hand, significantly increased C3a and C4a levels with filtration. In addition, there was no PF4 adsorption ability during the filtration. The filters for red cells (RC50, BPF4, and R500A) had no anaphylatoxin adsorption capabilities. The observed specific adsorption of anaphylatoxins might be attributed to the electrostatic force between the positively charged anaphylatoxins with high p1 and the possibly negatively charged filter membranes. Since PF4 and β‐TG have positively charged moieties in the C‐terminal position, the same adsorption mechanism might operate. We have obtained a useful scavenging filter for the evaluation of side effects of anaphylatoxins on patients who received old PCs.

Presence of Propionibacterium acnes in blood components

BACKGROUND: Sterility testing, as part of the QC of blood components at the Japanese Red Cross Aichi Blood Center between April 1998 and March 2000, showed that 10 of 5568 tested blood components (0.18%), all of which were RBC concentrates, were contaminated with bacteria. Nine isolates were Propionibacterium acnes and one was Staphylococcus capitis.

A new polyvinylchloride blood bag plasticized with less‐leachable phthalate ester analogue, di‐n‐decyl phthalate, for storage of platelets

To compare changes in platelets stored in the new di‐n‐decyl phthalate (DnDP)‐plasticized polyvinyl chloride (PVC) bag with those in a di‐(2‐ethylhexyl) phthalate (DEHP)‐plasticized PVC bag, single‐donor apheresis platelet concentrates (PCs), 133 ± 11 × 107 platelets per ml (n = 7), were stored with 94 ± 3 ml of plasma in a new 1‐liter bag with a surface area of 44 ± 7.1 cm2 per 1010 platelets. Oxygen and carbon dioxide gas diffusion properties of PVC‐DnDP films were respectively, 1.6 and 2 times those of standard PVC‐DEHP films. The amounts of DnDP leaked into the plasma of PCs were low at 0.58 ± 0.06 mg per bag after 5‐day storage, which is about one‐eightieth the amount of DEHP leaked. The pH of PCs in PVC‐DnDP bags amounted to 6.99 ± 0.03 after 5‐day storage, with glycolysis accelerated somewhat in the new bags. However, the platelet oxygen consumption was no different from that in the PVC‐DEHP bags. Platelet aggregation and responses to hypotonic shock were significantly better in the new bags at the end of storage. Shape changes of platelets into spherical forms with dendrites were more frequently observed in PVC‐DnDP bags than in PVC‐DEHP bags. The study indicated that platelets stored in the new DnDP‐plasticized PVC bags have retained aggregation and responses to hypotonic shock more than platelets in the PVC‐DEHP bags, but spherical forms and anaerobic metabolism increased in the new bags.