A prospective study of G-CSF effects on hemostasis in allogeneic blood stem cell donors.
(17/1660)
Granulocyte colony-stimulating factor (G-CSF) is used in healthy donors of peripheral blood stem cells (PBSC) for allogeneic transplantation. However, some data have recently suggested that G-CSF may induce a hypercoagulable state, prompting us to study prospectively 22 PBSC donors before and after G-CSF 5 microg/kg twice daily. We sought evidence for changes in the following parameters: platelet count, von Willebrand factor antigen (vWF:Ag) and activity (vWF activity), beta-thromboglobulin (beta-TG), platelet factor 4 (PF-4), platelet activation markers (GMP-140 and PAC-1), activated partial thromboplastin time (aPTT), prothrombin time (PT), coagulant factor VIII (FVIII:C), thrombin-antithrombin complex (TAT), prothrombin fragment 1+2 (F1+2), thrombomodulin (TM) and tissue plasminogen activator antigen (tPA:Ag) prior to G-CSF and immediately before leukapheresis. ADP-induced platelet aggregation studies were also performed. G-CSF administration produced only mild discomfort. We found a significant increase in vWF:Ag (from 0.99 +/- 0.32 U/ml to 1.83 +/- 0.69 U/ml; P < 0.001), in vWF activity (from 1.04 +/- 0.34 U/ml to 1.78 +/- 0.50 U/ml; P < 0.001) and in FVIII:C (from 1.12 +/- 0.37 U/ml to 1.73 +/- 0.57 U/ml; P < 0.001) after G-CSF. Of note, four donors with low baseline vWF had a two- to three-fold increase after receiving G-CSF. G-CSF had no impact on the platelet count, beta-TG, PF-4, GMP-140 or PAC-1. The final% of platelet aggregation decreased from 73 +/- 22% to 37 +/- 26% after G-CSF (P < 0.001). We found a significant decrease in aPTT after G-CSF (29.9 +/- 3.1 s to 28.3 +/- 3.3 s; P = 0.004), but the PT was unaffected. In addition, we also observed a significant increase in TAT, F1+2 and TM, but not in tPA:Ag. Our data suggest that G-CSF may possibly induce a hypercoagulable state by increasing levels of FVIII:C and thrombin generation. In contrast to this information, we found reduced platelet aggregation after G-CSF administration. The clinical implications of these findings remain unclear and larger studies are definitely required. (+info)
Expression of factor VIII by murine liver sinusoidal endothelial cells.
(18/1660)
Factor VIII (fVIII) is the procoagulant plasma glycoprotein that is missing or decreased in hemophilia A. The cellular origin of fVIII synthesis is controversial. Liver transplantation cures hemophilia A, demonstrating that the liver is a major site of fVIII synthesis. We detected fVIII mRNA in purified populations of murine liver sinusoidal endothelial cells (LSECs) and hepatocytes, but not Kupffer cells. LSECs and hepatocytes contained comparable numbers of fVIII mRNA (40 and 70 transcripts per cell, respectively) by quantitative competitive reverse transcriptase-polymerase chain reaction analysis. There was not detectable mRNA for factor IX, a hepatocyte marker, in the LSEC preparation, nor was there detectable mRNA for von Willebrand factor, an endothelial cell marker, in the hepatocyte preparation. This excludes the possibility that detectable fVIII mRNA is due to cross-contamination in the hepatocyte or LSEC preparations. Primary cultures of LSECs were established in which fVIII mRNA levels were indistinguishable from purified LSECs. LSECs secreted active fVIII into the culture medium. This finding represents the first demonstration of homologous expression of fVIII mRNA and protein in cell culture and should facilitate studies of fVIII gene regulation. Additionally, LSECs potentially are targets for a fVIII transgene during gene therapy of hemophilia A. (+info)
The relationship of fibrinogen and factors VII and VIII to incident cardiovascular disease and death in the elderly: results from the cardiovascular health study.
(19/1660)
Little is known about the prospective associations of fibrinogen, factor VII, or factor VIII with cardiovascular disease (CVD) and mortality in the elderly. At baseline in the Cardiovascular Health Study (5888 white and African American men and women; aged >/=65 years), we measured fibrinogen, factor VIII, and factor VII. We used sex-stratified stepwise Cox survival analysis to determine relative risks (RRs) for CVD events and all-cause mortality (up to 5 years of follow-up), both unadjusted and adjusted for CVD risk factors and subclinical CVD. After adjustment, comparing the fifth quintile to the first, fibrinogen was significantly associated in men with coronary heart disease events (RR=2.1) and stroke or transient ischemic attack (RR=1.3), and also with mortality within 2.5 years of follow-up (RR=5.8) and later (RR=1.7). Factor VIII was significantly associated in men with coronary heart disease events (RR=1.5) and mortality (RR=1.8), and in women with stroke/transient ischemic attack (RR=1.4). For both factors, values were higher in those who died, whether causes were CVD-related or non-CVD-related, but highest in CVD death. Factor VII exhibited associations with incident angina (RR=1.44) in men and with death in women (RR, middle quintile compared with first=0.66). However, in general, factor VII was not consistently associated with CVD events in this population. We conclude that, if confirmed in other studies, the measurement of fibrinogen and/or factor VIII may help identify older individuals at higher risk for CVD events and mortality. (+info)
A pathologic study of abdominal lymphangiomas.
(20/1660)
Abdominal lymphangiomas are uncommon angiomatous tumor occurring mainly in childhood. This is a retrospective clinicopathologic study of 17 cases of abdominal lymphangioma. The patients included are five children and 12 adults, with a mean age at initial presentation of 30.7 years (age ranges 3-63). The locations of the tumors were mesentery (5), retroperitoneum (4), colon (3), omentum (3), mesocolon (1) and gallbladder (1). Infiltrative growth was more common pattern than entirely circumscribed pattern. Masses were mostly multilocular cysts and contained chyle or serous fluid. On immunohistochemical staining, 16 cases were reactive for either CD31 or factor VIII-related antigen. These fact would suggest that intra-abdominal lymphangiomas simulate the immunohistochemical features of collecting lymphatics. Follow up was possible in 12 cases for 3-50 months (mean 19 months) and only one patient showed local recurrence. Although abdominal lymphangiomas are rare in adulthood and correct preoperative diagnosis is difficult, awareness of such a possibility in adulthood will contribute to make a correct preoperative diagnosis. (+info)
Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII.
(21/1660)
FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A. (+info)
The effect of the angiogenesis inhibitor TNP-470 on luteal establishment and function in the primate.
(22/1660)
Angiogenesis during luteal development is probably essential for normal lutein cell function. Since the angiogenesis inhibitor TNP-470 inhibits pregnancy in mice, the current study investigated its effects on the establishment and function of the primate corpus luteum. Regularly ovulating macaques were treated with TNP-470 (6 mg/kg), i.v. in three doses, 48 h apart. Serum progesterone concentrations, as indicators of treatment effect, were normal in four macaques where treatment commenced at the onset of the ovulatory progesterone rise, and in five of eight in which treatment commenced a few days before ovulation. In the other three the normal progesterone rise was absent. To investigate the direct effect on luteal angiogenesis of a daily dose over a longer period, four marmosets received 18 mg/kg/day of TNP-470 i.v. for 9 days starting at ovulation. On day 10, luteal cell proliferation was determined by nuclear bromodeoxyuridine incorporation. Luteal microvasculature was examined using immunocytochemical factor VIII staining, and endothelial cell and luteal function assessed by in-situ hybridization of insulin-like growth factor binding protein-3 mRNA and plasma progesterone concentrations respectively. None of these parameters were affected by the TNP-470 treatment. The results show that, with the treatment regimens employed, TNP-470 had no significant effect on the expression of the differentiated state of the primate corpus luteum. (+info)
The light chain of factor VIII comprises a binding site for low density lipoprotein receptor-related protein.
(23/1660)
In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K(d) approximately 60 nM). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC(50) approximately 1 nM). In addition, a 12-fold molar excess of the physiological carrier of FVIII, i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of (125)I-labeled FVIII by LRP-expressing cells ( approximately 8 fmol/10(5) cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k(on) approximately 5 x 10(4) M(-1) s(-1); k(off) approximately 2.5 x 10(-3) s(-1); K(d) approximately 50 nM). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on the in vivo survival of FVIII. (+info)
Effect of postmenopausal hormones on inflammation-sensitive proteins: the Postmenopausal Estrogen/Progestin Interventions (PEPI) Study.
(24/1660)
BACKGROUND: Observational studies in healthy women suggest postmenopausal hormone therapy reduces risk of coronary events. In contrast, in a recent clinical trial of women with coronary disease, a subgroup analysis demonstrated increased risk during the early months of therapy. Because higher levels of inflammation factors predict vascular disease outcomes, the effect of hormones on these factors is of interest. METHODS AND RESULTS: Four inflammation-sensitive factors, C-reactive protein, soluble E-selectin, von Willebrand factor antigen, and coagulation factor VIIIc were measured at baseline, 12, and 36 months in 365 participants of the Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial, a randomized, placebo-controlled trial of the effects of 4 hormone preparations on cardiovascular disease risk factors. Compared with placebo, all 4 active preparations resulted in a large sustained increase in the concentration of C-reactive protein and a decrease in soluble E-selectin (P=0.0001). There were no effects of treatment on concentrations of von Willebrand factor or factor VIIIc. There were no differences in effects among treatment arms. Relative to placebo, when combining active treatment arms, final concentrations of C-reactive protein were 85% higher whereas E-selectin was 18% lower compared with baseline. CONCLUSIONS: Postmenopausal hormones rapidly increased the concentration of the inflammation factor C-reactive protein. Such an effect may be related to adverse early effects of estrogen therapy. In contrast, hormones reduced the concentration of soluble E-selectin, and this might be considered an anti-inflammatory effect. Because PEPI was not designed to assess clinical endpoints, studies of the impact of hormone-mediated changes in inflammation on risk of subsequent coronary events are needed. (+info)