Function-modulating human monoclonal antibodies against platelet-membrane receptors isolated from a phage-display library.

Monoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbalpha binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies.

Changes in the effects of interleukin-1beta and tumor necrosis factor-alpha on platelet activation in early pregnancy.

Platelets are known to be activated in normal pregnancy, and are further activated in pathological pregnancy states, such as preeclampsia. The factors controlling platelet activation are unknown, but cytokines, such as interleukin 1beta (IL-1beta) and tumor necrosis-alpha (TNF-alpha) have been found to affect platelet function and are believed to be involved in early pregnancy. We assessed the effects of these cytokines on platelets from women at various stages of pregnancy. We compared two methods: platelet in vitro aggregation by aggregometry, and platelet P-selectin expression by flow cytometry. IL-1beta and TNF-alpha had no effect on the in vitro aggregation and P-selectin expression of platelets from women in the first trimester of pregnancy as compared to the inhibitory effects of both in late pregnancy. We conclude that maternal platelet function undergoes a marked change throughout pregnancy.

Regulation of platelet aggregation and adenosine triphosphate release in vitro by 17beta-estradiol and medroxyprogesterone acetate in postmenopausal women.

Clinical studies have suggested that hormone replacement therapy (HRT) may reduce the risk of coronary heart disease in postmenopausal women. Although progestins are commonly added to HRT preparations for uteroprotection, the perceived beneficial cardiovascular effects of HRT are thought to be mediated predominantly by the estrogen component. Platelets play a critical role in the pathogenesis of atherosclerosis and cardiovascular disease and, hence, it is possible that the cardiovascular effects of estrogens are mediated, at least in part, through inhibition of illicit platelet activation. The aim of this study was to examine the effects of sex steroids on adenosine diphosphate (ADP)-induced platelet aggregation and adenosine triphosphate (ATP) release in vitro in postmenopausal women. In addition, the effects of antiestrogens 14-hydroxy tamoxifen (4-OHT) and ICI 182780] and antiprogestins (RU 486 and ZK 98299) were also investigated. Preincubation of platelet-rich plasma (PRP) with antiestrogens or antiprogestins did not alter subsequent platelet aggregation or ATP release in response to ADP. However, preincubation with 17beta-estradiol (E2) significantly inhibited ADP-mediated platelet aggregation by a mean (+/-SEM) of 37%+/-6% (p = 0.02) and ATP release by 82%+/-6% (p = 0.03), an effect that was reversed by the addition of ICI 182780 or 4-OHT but not RU 486 and ZK 98299. Although the progestin medroxyprogesterone acetate (MPA) also significantly inhibited platelet aggregation (by 28%+/-5%, p = 0.02) and ATP release (by 63%+/-9%, p = 0.02), this inhibition was not reversed by the addition of antiprogestins or antiestrogens. These data show that sex steroids can modulate platelet function in vitro. Furthermore, as platelets are devoid of nuclear components, these findings indicate that estrogens may regulate platelet function through binding to a non-nuclear receptor with ligand-binding properties similar or identical to the wild-type receptor. By contrast, MPA appears to exert its effect through a mechanism that does not involve binding to the "classical" progesterone receptor.

Cutaneous necrosis as a terminal paraneoplastic thromboembolic event in a patient with non-Hodgkin's lymphoma.

Thrombotic complications in non-Hodgkin's lymphoma often originate in the large veins. We describe a patient with refractory advanced high-grade lymphoma who presented with the rare complication of extensive cutaneous necrosis due to thrombosis of dermal vessels; there was also a recent new peak of monoclonal IgM-kappa protein. Direct immunofluorescence demonstrated immune deposits with complement in the dermal vessel wall. Based on these observations and on published data, we suggest that these complexes were the trigger for the thrombotic events and that the monoclonal IgM acted as xenoreactive antibodies, initiating a cascade of events. The first step of this cascade was activation of the complement and the membrane attack complex, which caused secretion of IL-1 alpha by endothelial cells, followed by overexpression of tissue factor on the surface of the dermal vessel wall endothelium. Dermal vessel thrombosis was the final event in this cascade.

Increased operative bleeding during orthopaedic surgery in patients with type I Gaucher disease and bone involvement.

To aid clinicians in identifying patients with type I Gaucher disease who are at risk of excessive bleeding, we reviewed the coagulation parameters of six affected patients with bone involvement who underwent orthopaedic surgery at two centers, and of 22 patients under treatment at another, seven of whom had total splenectomy. All patients were of Jewish Ashkenazi origin. Among the latter group, prolonged prothrombin time was noted in 81%. Incidence of clotting factor deficiency were as follows: factor XI, 36.3%; V, 31.8%; VIII, 27.2%; IX, 13.6%; and XII, 27.2%. Most of the abnormalities occurred in the non-splenectomized patients. Two of the six orthopaedic surgery patients had excessive intraoperative and postoperative bleeding. One, who underwent spinal decompression had prolonged prothrombin time, and the other, who had total hip replacement, showed a deficiency of factor XI. The second patient's hemoglobin level was maintained with transfusion of fresh frozen plasma during contralateral hip arthroplasty five months later. We suggest that preoperative evaluation of clotting factors and replacement therapy may prevent excessive bleeding in patients with type I Gaucher disease.

Activated protein C resistance in pediatric inflammatory bowel disease.

BACKGROUND: There is evidence for a hypercoagulable state in inflammatory bowel disease (IBD), and small vessel thrombosis has been identified in the bowel of patients with Crohn's disease, suggesting thrombosis as a possible etiologic factor. Activated protein C (APC) resistance is the most common inherited disorder leading to thrombosis and accounts for 30% to 40% of episodes of idiopathic venous thrombosis. METHODS: The prevalence of APC resistance was studied in 23 patients with IBD (17 with Crohn's disease, 6 with ulcerative colitis) and in 11 control subjects with recurrent abdominal pain or celiac disease, using an APC resistance screening method. RESULTS: One patient with Crohn's disease had a positive screen result, two patients (one with Crohn's, one with ulcerative colitis) had borderline results, and results in all of the control subjects were normal. One patient with Crohn's disease had a history of a thromboembolic event but had a normal screen result. CONCLUSIONS: Activated protein C resistance does not seem to play a major role in the etiology of the hypercoagulable state in inflammatory bowel disease.

Levels of proteins C and S do not decline subsequent to first line chemotherapy in lymphoma patients.

Thromboembolic complications and decrease in protein C and S have been observed in patients while receiving combination chemotherapy for breast cancer. We investigated whether initial cytotoxic treatment of non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) is also associated with changes in these anticoagulant parameters. For this purpose 25 patients with intermediate to high grade NHL and seven with HD, undergoing primary treatment with cytotoxic drugs were evaluated at three time-points: pre-therapy, mid-therapy and post-therapy. In contrast to the breast cancer patients, no significant changes in protein C, protein S and antithrombin III levels were observed in the NHL patients during the various stages of therapy. However in HD patients, the mean protein C values had a tendency to be higher at mid-therapy compared to pre-therapy and protein S levels had a tendency to be higher at mid-therapy compared to post-therapy. In lymphoma patients receiving primary cytotoxic treatment we did not find changes in anticoagulant parameters that can explain a chemotherapy-induced hypercoagulable state, as has been reported in breast cancer patients.

A human monoclonal IgA autoantibody--185/12--behaves like an autoimmune antiphospholipid antibody.

A human monoclonal anticardiolipin autoantibody (ACA) of the IgA-k isotype, designated 185/12, is described. The antibody was prepared from peripheral B cells, obtained from a patient with a history of habitual abortion, by immortalization with Epstein-Barr virus (EBV). The antibody displays a strong binding activity to cardiolipin and phosphatidyl L-serine, but not to phosphatidylcholine, phosphatidylinositol, ssDNA and dsDNA. It binds to cardiolipin in a concentration-related and saturable manner (Kd = 3.0 x 10(-8) M). This reaction is dependent upon the presence of bovine serum, and is fully inhibited by cardiolipin vesicles. The 185/12 antibody exhibits different binding patterns to the solid-phase bound cardiolipin-serum complex and to its individual components (cardiolipin and bovine serum). The Bmax of 185/12 binding to the complex (0.968 OD units) is higher than the sum of the Bmax values calculated for each one of the complex components (0.352 + 0.179 = 0.531 OD units). Bovine serum as well as purified beta 2-glycoprotein I (beta 2-GPI) in suspension inhibit the binding of 185/12 to the complex. 185/12 binding capacity increases in direct relation to the rising concentration of beta 2-GPI. Collectively, these data may be interpreted to suggest that 185/12 antibody, which is an IgA isotype, exhibits characteristics usually attributed only to antiphospholipid autoantibodies (APA) of the IgG isotype, that are associated with the clinical spectrum of APA syndrome (APA-S). It is, therefore, possible that autoantibodies of the IgA isotype could play a pathogenic role, which may be different from that of the IgG isotype, in the development of autoimmune phenomena.

The functions of thrombospondin and its involvement in physiology and pathophysiology.

The thrombospondin family of molecules is expressed in many different tissues. Its expression is highly regulated by different hormones and cytokines and is developmentally controlled. It can bind to many different cell types, probably via an array of receptors which are similarly regulated. The level of thrombospondins in body fluids and their distribution in tissue change in correlation with various pathological states. It is linked to the growth of primary tumors and to metastasis, to development of the atherosclerotic plaque, to malaria infection and other diseases. The role(s) of thrombospondin(s) are by and large unknown, though specific interaction seem to affect particular cell functions. The wide-spread spatial and temporal regulation, multiple interactions and correlation with major diseases imply important roles in cell function and call for concerted effort to unravel the mystery.

Differentiation-controlled synthesis and binding of thrombospondin to granulosa cells.

Thrombospondin (TSP) is a large glycoprotein, synthesized by several matrix-forming cells and incorporated into their extracellular matrix. In several cell types its presence supports cell growth and proliferation. To investigate the role of this protein in cell differentiation, we studied the hormonal effect of TSP production and receptor-mediated binding to primary granulosa cells prepared from diethylstilbestrol-treated immature female rats. These cells can be induced to differentiate by FSH, 8-bromo-cAMP (8-Br-cAMP), or forskolin. Progesterone production is induced during differentiation, and its level of synthesis is an important manifestation of the differentiated phenotype. We find that undifferentiated granulosa cells synthesize and secrete TSP. The protein comprises about 0.5% of the total cell protein, and it is the major protein secreted in culture. Treatment of the cells with FSH or 8-Br-cAMP reduces TSP production dramatically, and forskolin completely inhibits it. In parallel, we observed that the undifferentiated cells bind TSP specifically with a Kd of 1.8 nM, and the number of binding sites per cell is 1.7 x 10(5). This binding can be prevented by excess TSP or an anti-TSP monoclonal antibody (B7-3). This ability to bind TSP is completely lost after induction of differentiation by FSH or 8-Br-cAMP. Our findings show that both the production and binding of TSP to granulosa cells are tightly controlled by normal cell differentiation and indicate that changes in TSP are correlated with the passage of the cell through the stages of maturation, a passage that also involves changes in cell shape and extracellular interactions and in the steroidogenic capacity of these cells.

Gray platelet syndrome in the elderly.

A 68-year-old male who suffered from thrombocytopenia and mild splenomegaly for 18 years was found to present agranular gray platelets on peripheral blood smear. Bone biopsy revealed a mild, diffuse, reticular fibrosis with no collagen, and electron microscopy of the platelets showed an absence of almost all the alpha-granules. Platelet thrombospondin and fibronectin analysed by SDS-polyacrylamide gel electrophoresis and Rocket immunoelectrophoresis were absent. Follow-up of 4 years showed the same parameters with no evidence of active myeloproliferative or dysmyelopoietic disorders. Hemorrhagic diathesis was limited to ecchymoses and postprostatectomy bleeding, necessitating platelet transfusion. This led us to conclude that our patient probably had a constitutional primary alpha-granule deficiency or gray platelet syndrome. This extremely rare defect has been described in less than 10 patients, all of them very young. Our observation shows that these patients may have a long, uneventful survival.