Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates

Thrombopoietin (TPO) is the physiologic Mpl-ligand regulating platelet production. Pegylated human recombinant megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated polypeptide Mpl-ligand derivitized with poly-(ethylene glycol), induces megakaryocyte endoreduplication and proliferation in vitro and in vivo. In the present study, the dose-response effects of PEG-rHuMGDF on pharmacokinetics, megakaryocytopoiesis, platelet production, and platelet function were characterized for dosing 0.05, 0.10, 0.50, or 2.5 micrograms/kg/d in 22 baboons for 28 days. Daily subcutaneous injections of PEG-rHuMGDF produced linear log-dose responses in (1) steady-state trough plasma levels of PEG-HuMGDF (P < 10(-3)); (2) marrow megakaryocyte volume (P < 10(-3)), ploidy (P < 10(-4)), and number (P < .01); and (3) peripheral platelet concentrations (P < 10(- 4)) and platelet mass turnover (P < 10(-3)). Platelet morphology, life span, and recovery were normal, and peripheral leukocyte, neutrophil, and erythrocyte counts were not significantly affected by PEG-rHuMGDF (P > .1 in all cases). PEG-rHuMGDF at 0.5 micrograms/kg/d produced similar blood concentrations of Mpl-ligand and platelets as 10 times the dose of rHu-MGDF (5.0 micrograms/kg/d), reflecting the extended plasma half-life achieved through pegylation. Whereas PEG-rHuMGDF did not induce platelet aggregation in vitro, platelet aggregatory responsiveness induced by thrombin receptor agonist peptide (TRAP1-6) and collagen was transiently enhanced ex vivo during the initial few days of PEG-rHuMGDF administration. However, adenosine diphosphate (ADP)-induced platelet aggregation was not enhanced ex vivo by PEG- rHuMGDF therapy. 111In-platelet deposition on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts increased in direct proportion to the circulating platelet concentration (P < 10(-4) for both EA and VG); 125l-fibrin accumulation was not affected by PEG-rHuMGDF-induced increases in peripheral platelet counts. Changes in platelet production and function produced by PEG-rHuMGDF returned to baseline within 2 weeks after discontinuing treatment. Thus, in nonhuman primates, PEG- rHuMGDF increases platelet production in a linear log-dose-dependent manner by stimulating megakaryocyte endoreduplication and new megakaryocyte formation from marrow hematopoietic progenitors. These findings suggest that appropriate dosing of PEG-rHuMGDF therapy during periods of chemotherapy-induced marrow suppression may maintain hemostatic concentrations of peripheral platelets without increasing the risk of thrombosis.     

Demonstration of single chain urokinase-type plasminogen activator on human platelet membrane

Fibrinolytic activity was found to be associated with sonicated platelet membranes after separation from cytosol by differential centrifugation. This fibrinolytic activity was attributed to the presence of a plasminogen activator, which was immunochemically identified as urinary-type plasminogen activator (uPA) by antibody neutralization assay, immunoblotting, and immunofluorescence. The molecular weight (mol wt) of this uPA was 54,000 and was present as the single chain form, although a small amount was detected in a higher mol wt complex indicative of a uPA-inhibitor complex. Treatment of membrane preparations with Triton X-100, 3 mol/L KCl, and 0.1 mol/L glycine, (pH 2.3), but not 10 mmol/L ethylenediamine tetraacetic acid (EDTA), removed the uPA from the membrane. This suggests that uPA is a peripheral membrane protein and that metal ions do not mediate protein- membrane association. Immunofluorescent staining revealed the presence of uPA on the outer surface of the platelet in preparations of intact unstimulated platelets. Thus, uPA is associated with the outer leaflet of the platelet membrane and may be involved with the acceleration of thrombus degradation observed with platelet-rich thrombi.     

Nipradilol, a new β-blocker with vasodilatory properties, in experimental portal hypertension: A comparative haemodynamic study with propranolol

The haemodynamic effects of nipradilol, a new non-selective β-adrenoreceptor blocker with vasodilating actions like nitroglycerin, were examined in rats with portal hypertension due to portal vein stenosis. Portal hypertensive rats were divided into five groups receiving infusion of placebo, 3 mg of propranolol, 300, 600 and 1200 μg of nipradilol. At its highest dose, nipradilol achieved a reduction of 34.4 ± 4.4% in heart rate which was similar to that in the propranolol group (36.5 ± 2.4%). Also for other systemic haemodynamic parameters, the nipradilol 1200 μg group exhibited changes not significantly different from those in the propranolol group; mean arterial pressure (- 13 vs - 14%), cardiac index (- 37 vs - 31%) and systemic vascular resistance (+ 29 vs+ 32%). In contrast to the similar changes in the systemic circulation, a 1200 μg dose of nipradilol lowered portal pressure significantly more than propranolol (- 4.3 ± 0.6 vs - 2.9 ± 0.2 mmHg, P≤ 0.05). Nipradilol then reduced portal blood flow by 22% (P≤ 0.05) without a significant change in portocollateral resistance. On the other hand, propranolol not only caused a reduction in portal blood flow of 30% (P≤ 0.01), but also an increase in portocollateral resistance of 21% (P≤ 0.05). The results suggest that nipradilol may ensure a more effective control of portal hypertension than propranolol, presumably via its venodilatory action on portocollateral vessels.     

CD63 is a component of Weibel-Palade bodies of human endothelial cells

Weibel-Palade bodies are secretory granules of vascular endothelial cells specialized in the storage of von Willebrand factor (vWF) and P- selectin, two adhesion proteins that can be rapidly mobilized to the cell surface by exocytosis in response to thrombin or other agonists. In this study, we attempted to identify additional components of Weibel- Palade bodies by raising monoclonal antibodies to these granules, purified by cell fractionation. One antibody, 2C6, was found to be specific for CD63, a membrane glycoprotein previously described in the lysosomes of platelets and other cell types. The immunopurified 2C6 antigen was recognized by an anti-CD63 reference antibody, 2.28, by Western blotting. Also, the biosynthetic profile of the 2C6 antigen in endothelial cells showed a nascent molecular mass and a glycosylation pattern identical to that of CD63. Immunofluorescence staining with 2C6 showed the lysosomes, and also elongated structures identified as Weibel-Palade bodies by their shape, distribution, and positive staining with anti-vWF antibodies, CD63 was also found by Western blotting of subcellular fractions highly enriched in Weibel-Palade bodies. Our results indicate that CD63 colocalizes with vWF and P- selectin in the Weibel-Palade bodies of endothelial cells, and together with these adhesion proteins it could be rapidly expressed on the cell surface in areas of vascular injury and inflammation.     

What harm does a second delivery to the pelvic floor?

Objective

To compare the pelvic floor function of primiparous women to women after a second delivery regarding symptoms of urinary and anal incontinence, anal sphincter ruptures and bladder-neck mobility.

Methods

A questionnaire evaluating symptoms of urinary and anal incontinence was used in nulliparous women before and 27 months after childbirth. Furthermore these symptoms were correlated with functional changes of the pelvic floor based on a careful gynecologic examination as well as perineal and endoanal ultrasound.

Results

112 nulliparous women were included, 49 women returned for follow-up on average 27 months (SD 4.4 months) after the first delivery. 39 women (group A) had just one delivery, 10 women (group B - 10/49) had had a second delivery. Apart from levator ani muscle strength, no significant difference between pelvic floor function of group A vs group B was demonstrable. Furthermore, we could show no significant difference for symptoms of urinary (11 (28.2%) vs. 5 (50.0%)) and anal incontinence (14 (35.9%) vs. 4 (40.0%)) between both groups. However, we found a lasting increase of stress urinary and anal incontinence as well as overactive bladder symptoms after one or more deliveries. The position of the bladder neck at rest was lower in both groups compared to the position before the first delivery and bladder neck mobility increased after one or more deliveries.

Discussion

Our study shows several statistically significant changes of the pelvic floor function even on average 27 months after delivery, but a subsequent delivery did not compromise the pelvic floor any further.     

Flow cytometric detection of tumor metastases during urologic surgery

Flow cytometric studies were performed on peripheral blood obtained before, during, and subsequent to urologic surgery. The results suggest that 7 of 11 patients had a brief period during surgical manipulation when they had cells from the primary surgical site in the peripheral blood. Concentration of such cells was generally less than 10(2)/mL even during the intraoperative period. These findings suggest that perioperative tumor seeding is a real concern, and that flow cytometry can be used to study maneuvers to reduce its incidence.     

Pulmonary hamartoma: diagnosis by transthoracic needle-aspiration biopsy

Hamartomas of the lung often present as asymptomatic, noncharacteristic masses that can seldom be differentiated from other lung masses such as primary cancer or metastases by conventional radiography. Transthoracic needle-aspiration biopsy (TNAB) has become a popular and reliable method for the diagnosis of a lung lesion, and it offers a valuable alternative to diagnostic thoracotomy. In our study, TNAB established the diagnosis of pulmonary hamartoma in 12 of 14 (86%) patients. In eight patients, one procedure (using one to three punctures) was sufficient to establish the diagnosis, and, in four patients, two procedures (using one to two punctures) were necessary. In two patients, the lesion was missed on second and/or third biopsy procedures, and the correct diagnosis was obtained at surgery. Cytologic examination of the material was diagnostic in five of the 14 patients. Tissue specimens were sent in 13/14 patients, and findings of histologic examination established the correct diagnosis in 11 of these patients. One of the 12 patients in whom hamartoma was correctly diagnosed by TNAB underwent resection of his lesion, and histologic examination confirmed the diagnosis. In the remaining 11 patients, the lesions have been stable on follow-up chest examinations.