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.     

Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.     

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.     

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.     

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.     

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.     

Renal responses to atrial natriuretic peptide are preserved in bilateral ureteral obstruction and augmented by neutral endopeptidase inhibition

PURPOSE: Atrial natriuretic peptide (ANP) contributes to post-obstructive diuresis in bilateral ureteral obstruction (BUO). In this study we examined the activity of neutral endopeptidase (NEP), an enzyme responsible for degradation of ANP, in the kidney in rats subjected to BUO for 24 hours. MATERIALS AND METHODS: Renal function was examined by the clearance method in sham operated rats and BUO rats after obstruction release. Renal responses to an intravenous bolus injection of ANP (5 microg/kg) were studied in sham operated and BUO rats with or without pretreatment with intravenous phosphoramidon (100 microg/kg per minute), a NEP inhibitor. RESULTS: In BUO rats natriuresis and diuresis occurred despite a marked decrease in the glomerular filtration rate (GFR). ANP administration increased GFR and induced marked natriuresis and diuresis in sham operated and BUO rats. Inhibition of ANP degradation by phosphoramidon induced natriuresis and diuresis, and accentuated these renal responses to ANP. CONCLUSIONS: Renal responses to ANP and renal NEP activity were preserved in 24-hour BUO. NEP inhibition to attenuate ANP degradation augmented responses to ANP in increasing GFR, natriuresis and diuresis. These findings provide the theoretical potential for facilitating the recovery of GFR after BUO release by inhibiting ANP degradation by pharmacological means.