M. Yanabu

Relationship of microparticles withβ2-glycoprotein I and P-selectin positivity to anticardiolipin antibodies in immune thrombocytopenic purpura

We investigated the association ofβ2-glycoprotein I and P-selectin with platelet-derived microparticles in 48 patients with immune thrombocytopenic purpura and 20 normal controls using two-color flow cytometric analysis. In addition, anticardiolipin antibodies were detected by an enzyme-linked immunosorbent assay. Platelet microparticles from the patients showed a higher positivity forβ2-glycoprotein I than those from the normal controls (23.1±15.4% vs. 5.3±3.1%, p<0.01), but this positivity was not related to the presence of platelet-associated IgG or to the severity of thrombocytopenia. In the 18 patients with more than 20% P-selectin-positive microparticles,β2-glycoprotein I positivity was significantly higher than in the 30 patients with less than 20% P-selectin-positive microparticles (37.1±20.5% vs. 21.5±17.3%, p<0.01). In addition, anticardiolipin antibodies were detected in eight patients, and they had a significantly higher level ofβ2-glycoprotein I-positive microparticles than the patients without such antibodies (42.0±22.9% vs. 22.6±18.9%, p<0.05). Our results suggest that anticardiolipin antibodies activate platelets in immune throm bocytopenic purpura and cause the generation of microparticles rich inβ2-glycoprotein I and P-selectin. These microparticles may then act to regulate coagulation abnormalities in patients with anticardiolipin antibodies.

Anti-phospholipid antibodies bind to platelet microparticles in idiopathic (autoimmune) thrombocytopenic purpura.

SummaryWe recently reported that IgM antibody-related microparticles exist in some patients with idiopathic thrombocytopenic purpura (ITP) [14]. In this study, we investigated the relationship between antiphospholipid (cardiolipin and phosphatidylinositol) antibodies and microparticles in 56 ITP patients. We used an ELISA to detect anti-phospholipid antibodies. IgG antibodies against cardiolipin and phosphatidylinositol were detected in 13 and 12 patients, respectively. The titers of IgG antibodies against these phospholipids did not correlate with the platelet-associated IgG level or the platelet count. Next, we investigated the binding of anti-phospholipid antibodies to platelets and microparticles. Microparticles were obtained by incubating washed platelets with collagen plus thrombin. ITP plasma containing IgG-class anti-phospholipid antibodies showed significantly increase binding to microparticles compared with plasma without such antibodies (p<0.001). Our results suggest that anti-phospholipid antibodies could affect the function of platelet microparticles in ITP.

New monoclonal anti‐human Fc gamma receptor II antibodies induce platelet aggregation

We developed two new monoclonal antibodies, designaled NNKY3‐2 and NNKY4‐7, that recognized a 40‐kD platelet protein. They appeared to be monoclonal anti‐Fc gamma receptor II (FC‐/RII) antibodies from the results of flow cytometric binding inhibition studies using another monoclonal anti‐FcγRII antibody (2E1). The addition of NNKY3‐2 or NNKY4‐7 to platetet‐rich plasma (PRP) led to a typical aggregation pattern preceded by a lag phase, but their addition to washed platelets did not induce aggregation. The aggregation of PRP by these antibodies was inhibited by prostaglandin E1 (PGE1) or staurosporine (protein kinase C inhibitor), whereas it was only slightly affected by a monoclonal anti‐GPIIb/IIIa antibody or Arg‐Gly‐Asp‐Ser, Furthermore, these antibodies induced the aggregation of washed platelets plus normal serum, but not that of washed platelets plus heat‐treatcd serum (destruction of complement activity). These results suggest that NNKY3‐2 or NNKY4‐7‐induced aggregation involves an unusual pathway independent of fibrinogen, and that the important factor is the participation of complement. NNKY3‐2 and NNKY4‐7 may be useful to study the relationship between autoantibodies, the Fc receptor, and complement in idiopathic thrombocytopenic purpura.

Characterization of a Novel Activation-Specific Antiplatelet Monoclonal Antibody

We developed a murine monoclonal antibody (KmT-50) which binds to a protein expressed on the surface of human platelets after activation. KmT-50 is an immunoglobulin G1 monoclonal antibody that recognizes a 50-kD antigen localized in intracellular organelles and the open canalicular system of unstimulated platelets. After platelets were stimulated, the antigen was secreted via the surface-connected canalicular system and exposed on the platelet membrane. KmT-50 bound not only to human platelets, but also to rabbit and monkey platelets. This new antibody may be useful for experimental studies on thrombosis and hemostasis.

Hypertension and diabetes mellitus increase the risk of haemorrhage in chronic idiopathic thrombocytopenic purpura.

The risk factors for haemorrhage in chronic idiopathic thrombocytopenic purpura (ITP) remain poorly understood. We classified 49 patients with chronic ITP into two groups on the basis of the presence (n = 11) or absence (n = 38) of hypertension and/or diabetes mellitus, and then analyzed their clinical and immunological characteristics. The patients with hypertension and/or diabetes were older than those without these complications. There were no significant differences between the two groups with regard to platelet count or the levels of platelet-associated immunoglobulin G, platelet-associated immunoglobulin M, and platelet-associated C3. Positivity for anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib autoantibodies was also similar. However, severe purpura and a poor response to prednisolone were far more common in the patients with hypertension and/or diabetes. We conclude that ITP complicated by hypertension and/or diabetes may be resistant to prednisolone and thus require more careful treatment.

Platelet Transfusion for Patients with Glanzmann's Thromboasthenia

We read with interest the recent paper by Ito et al. [l] on antibody removal therapy in a patient with Glanzmann’s thromboasthenia (GT). They described a successful antibody removal therapy at delivery in a patient with GT who had multiple antiHLA antibodies and an anti-glycoprotein (GP) IIb/IIIa antibody. GT is characterized by a bleeding tendency associated with abnormalities of GP IIb/IIIa. Even in patients with GT, if they have been bleeding severely, circumstances may allow therapy with transfusion of platelets. However, such patients may acquire antibodies against platelet GP IIb/IIIa. We recently attempted transfusion of platelets in a patient with GT who had severe and progressive anemia due to genital bleeding. Consequently, her genital bleeding stopped. However, the same symptoms reappeared after a few months. We again attempted transfusion of platelets, but this time bleeding persisted. On day 3 after transfusion, the serum completely inhibited aggregation of normal platelets upon ADP stimulation. Furthermore, it also blocked the binding of monoclonal anti-GP IIb/IIIa antibody to normal platelets using flow cytometric analysis. Thus, the patient seemed to have acquired antibodies against GP IIb/ IIIa. Alloimmune and isoimmune antibodies produced in patients with GT who have received multiple transfusions have been reported previously [2]. We think that platelet transfusion for patients with GT should be administered carefully. However, when the patient’s life is in peril, platelet transfusion may be necessary. The report by Ito et al. [l] provides a useful insight into the prognosis of such patients. .............................................. References

Activation of transfused platelets in a patient with amegakaryocytic thrombocytopenic purpura

(on average) 260 mL and 125 WBCs per pL (32 x lo6 WBCs/ unit). Analysis of WBCs by flow cytometry yielded 46 percent lymphocytes, including 3 1 percent CD4-positive T-lymphocytes (mean, 6 units). In all nonfrozen plasma samples, the lymphocytes exhibited growth capacity, detected by the lymphocyte transformation test. Using the stem cell assay, hematopoietic progenitor cells showed colony-forming capacity; plasma bags contained on average 5680 colony-forming units per 260 mL. After freezing and thawing, roughly 30 percent of the mononuclear cells remained intact, as determined by acridine orange staining. However, the lymphocytes no longer showed phytohemagglutinin-inducible proliferation capacity