Binding and Release of Factor VIII/von Willebrand''s Factor by Human Endothelial Cells

The uptake and release of factor VIII/von Willebrand's protein by cultured human umbilical vein endothelial cells have been examined using highly purified 125I-factor VIII possessing von Willebrand's factor activity. 125I-factor VIII/vWF was taken up by the cells, reaching maximum binding within 4 h with a t1-2 of binding of 15 min. Endothelial cell binding of 125I-factor VIII/vWF reached saturation at a concentration of 1.5 mg/l. Binding was inhibited by coincubation of excess unlabelled factor VIII/vWF. Most of the cell-associated radioactivity was released by treatment of the cells with trypsin. Internalization of bound protein was evidenced by the incorporation into the cells of radioactivity which could not be released by trypsin. Human vascular smooth muscle cells did not bind 125I-factor VIII/vWF. Addition of 0.1 microM epinephrine to the 125I-factor VIII/vWF labelled endothelial cultures induced the release of cell bound, protein-associated radioactivity into the medium. Propranolol inhibited completely epinephrine-induced release, whereas phenylephrine had no effect. Endothelial cells maintained in medium partially depleted of factor VIII/vWF by tricalcium citrate cellulose treatment of plasma did not release factor VIII antigen into the culture medium during subsequent incubation. Although [3H]proline was incorporated into proteins released by endothelial cells under these experimental conditions, specific incorporation of label into factor VIII/vWF antigen was not detectable by a sensitive solid-phase immunoradiometric assay. We conclude that factor VIII/vWF binds to endothelial cells and that this cell-bound protein is mobilized by epinephrine through beta-adrenergic stimulation.     

The combined use of monoclonal antibody- based enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII: Ag) and von Willebrand factor antigen (vWF: Ag) for the detection of carriers of haemophilia A

Enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII: Ag) and von Willebrand factor antigen (vWF: Ag) have been developed, each employing monoclonal antibodies. In the majority of severe haernophilic plasmas tested, VIII: Ag was undetectable by ELISA and also by immunoradiometric assay (IRMA) using haemophilic VIII:C antibodies. In haemophilic plasmas with mild/moderate deficiency of coagulant factor VIII (VIII: C), there was no significant difference between the two immunoassays although there was a general trend for ELISA VIII: Ag results to be higher. Assay of von Willebrand's disease (vWd) plasmas with the ELISA for vWF: Ag demonstrated reduced levels of this antigen in type I vWd, normal levels in type IIA, and a severe reduction of vWF:Ag in type III vWd. The discrimination of obligate carriers of haemophilia from normal was determined using ratios of factor VIII/vWF. Factor VIII antigen/von Willebrand factor antigen measured by IRMA and Laurell immunoelectrophoresis respectively, gave a superior discriminant to that of VIII: C/vWF: Ag (Laurell), but optimal discrimination was obtained with the combination of ELISAs for VIII: Ag and vWF: Ag.     

The combined use of monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII:Ag) and von Willebrand factor antigen (vWF:Ag) for the detection of carriers of haemophilia A

Enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII:Ag) and von Willebrand factor antigen (vWF:Ag) have been developed, each employing monoclonal antibodies. In the majority of severe haemophilic plasmas tested, VIII:Ag was undetectable by ELISA and also by immunoradiometric assay (IRMA) using haemophilic VIII:C antibodies. In haemophilic plasmas with mild/moderate deficiency of coagulant factor VIII (VIII:C), there was no significant difference between the two immunoassays although there was a general trend for ELISA VIII:Ag results to be higher. Assay of von Willebrand's disease (vWd) plasmas with the ELISA for vWF:Ag demonstrated reduced levels of this antigen in type I vWd, normal levels in type IIA, and a severe reduction of vWF:Ag in type III vWd. The discrimination of obligate carriers of haemophilia from normal was determined using ratios of factor VIII/vWF. Factor VIII antigen/von Willebrand factor antigen measured by IRMA and Laurell immunoelectrophoresis respectively, gave a superior discriminant to that of VIII:C/vWF:Ag (Laurell), but optimal discrimination was obtained with the combination of ELISAs for VIII:Ag and vWF:Ag.     

Sinusoidal endothelial cells from guinea pig liver synthesize and secrete cellular fibronectin in vitro

Endothelial liver cells were obtained from guinea pig by enzymatic digestion and centrifugal elutriation. Cells were cultured on gelatin and fibronectin pretreated culture vessels. Endothelial cells were characterized by phase-contrast microscopy, electron microscopy and the presence of Factor VIII-related antigen. Fibronectin secretion was determined in cell-free supernatants by a sensitive and specific ELISA and localized on fixed cultured cells by immunofluorescence. [35S]Methionine endogeneously labeled fibronectin was immunoprecipitated from supernatants and cellular lysates and displayed on sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. After attachment to the culture vessel, one day after plating, endothelial cells start to produce fibronectin as measured by ELISA and demonstrated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Secretion of fibronectin increases as cells proliferate to form a confluent monolayer. By immunofluorescence, fibronectin is visualized inside permeabilized cells and as a fibrillar network on the cell surface. Underneath the cell bodies, fibronectin-positive material is present as short strands. From supernatants and cellular lysates, fibronectin is immunoprecipitated with an apparent Mr of about 235,000 obviously larger than plasma fibronectin with an Mr of 220,000, which behaves electrophoretically like fibronectin isolated from early hepatocyte cultures. As endothelial cells incorporate [3H]fucose in fibronectin, whereas hepatocytes do not, we conclude that endothelial cells in contrast to hepatocytes produce cellular fibronectin. Endothelial cells, therefore, are probably the cellular source of the fibronectin present in the space of Disse. The significance of this finding with respect to fibrotic liver disease is discussed.     

Two novel type 2N von Willebrand disease-causing mutations that result in defective factor VIII binding, multimerization, and secretion of von Willebrand factor

Two novel mutations, a T-to-C transition at nucleotide 2612 and a T-to-G transversion at nucleotide 3923 of the von Willebrand factor (vWF) complementary DNA, were detected by analysis of the vWF gene in DNA from members of 2 families with atypical von Willebrand disease. The T2612C transition predicts substitution of cysteine by arginine at amino acid position 788 (C788R), and the T3923G transversion predicts substitution of cysteine by glycine at position 1225 (C1225G) of pre-pro-vWF. The patients homozygous for the C788R and C1225G mutations both had a partial vWF deficiency (0. 18 IU/mL and 0.07 IU/mL vWF antigen, respectively); vWF in plasma from patients homozygous for either the C788R or the C1225G mutation failed to bind factor VIII and lacked high molecular weight multimers. Recombinant (r) vWF molecules having the C788R or C1225G mutation were expressed in COS-7 cells. Both rvWF C788R and rvWF C1225G exhibited significantly impaired secretion and failed to bind factor VIII. Recombinant vWF C788R in COS-7 culture medium showed a severe reduction in high molecular weight multimers, whereas rvWF C1225G showed a very mild reduction in high molecular weight multimers when compared with wild-type rvWF. (Blood. 2000;95:2000-2007)     

Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.     

The effect of cultured endothelial cells on factor VIII procoagulant activity.

Cultured human umbilical vein endothelial cells produce a protein that has von Willebrand factor activity and forms immunoprecipitates with rabbit antibody to purified plasma factor VIII/von Willebrand factor (FVIII/vWF) protein, but it has no FVIII procoagulant activity. Of the three characteristics of plasma FVIII/vWF protein, only FVIII procoagulant activity is readily destroyed by trace proteases. A previous report from this laboratory demonstrated protease activity in culture medium under conditions that had been used by others to show that endothelial cells do not synthesize protein with FVIII procoagulant activity. However, even if cultured endothelial cells are placed in protease-free culture medium, no FVIII procoagulant activity can be detected, despite an increase in the level of protein with vWF activity from 0 to 0.57 microgram/ml by 48 hr. This observation and the lack of protease activity in medium left in contact with the cells for 48 hr led to the hypothesis that proteases exist on the surface of cultured umbilical vein endothelial cells. Protease activity was quantitated by the hydrolysis of p-nitroaniline from the substrate, N-benzoyl-phenylalanyl-valyl-arginyl-p-nitroanilide and by degradation of the procoagulant activity of added purified plasma FVIII/vWF protein. In the absence of endothelial cells, no protease activity was present in protease-free culture medium whether or not it had previously overlaid cultured cells. This medium did not cause cleavage of p-nitroaniline from the tripeptide substrate, and 83% of added FVIII procoagulant activity remained after 48 hr. When the synthetic tripeptide was incubated in contact with cultured endothelial cells, 7.3 +/- 0.8 X 10(-10) moles of p-nitroaniline/hr was released; moreover, only 47% of the added FVIII procoagulant activity remained after 48 hr. Given this rate of destruction, it can be calculated that sufficient protease activity exists on the surface of cultured endothelial cells to degrade the procoagulant activity of approximately 1.6 microgram FVIII/vWF protein/hr. This degradation rate is 45 times the rate of release of FVIII/vWF protein from cultured endothelial cells when assessed by the generation of protein with vWF activity. Hence, the detection of FVIII procoagulant activity, if in fact synthesized by cultured endothelial cells, will be most difficult.     

bFGF和PDGF-BB对内皮祖细胞分化的内皮样细胞和平滑肌样细胞增殖和迁移的影响

目的探讨内皮祖细胞分化的内皮样和平滑肌样细胞在碱性成纤维生长因子和血小板源生长因子BB作用下的增殖和迁移能力。方法将分离、培养及纯化的内皮祖细胞培养5天后进行分组:对照组、碱性成纤维生长因子组和血小板源长因子BB组。对照组使用20%胎牛血清的DMEM培养基;碱性成纤维生长因子组和血小板源生因子BB组在20%胎牛血清的DMEM培养基中分别添加碱性成纤维生长因子(30μg/L)和血小板源生长因子BB(40μg/L)。免疫荧光染色鉴定内皮祖细胞以及检测内皮祖细胞内皮方向分化标记物CD31和vWF,及平滑肌方向分化标记物α-SMA和Calponin。将分化的内皮样细胞和平滑肌样细胞用MTT法和Transwell小室分别检测其增殖活性和迁移能力。结果与对照组比较,内皮祖细胞经碱性成纤维生长因子或血小板源生长因子BB诱导后呈现较强的内皮细胞(CD31,vWF)或平滑肌细胞(α-SMA,Calponin)的荧光染色,被诱导细胞分别称为内皮样细胞和平滑肌样细胞;碱性成纤维生长因子促进内皮样细胞和血小板源生长因子BB促进平滑肌样细胞增殖的作用在一定范围内具有时间(0～48 h)和浓度(碱性成纤维生长因子:0～16μg/L;血小...     

Isolation of small molecular forms of Factor VIII/von Willebrand factor from plasma

Cryoprecipitated factor VIII/von Willebrand factor (FVIII/vWF), freed of fibrinogen by clotting with calcium and Defibrase, was chromatographed on Sepharose CL-2B. Fractions containing lower-molecular-weight forms of FVIII/vWF comprised coprecipitated plasma proteins of similar molecular weights. The major contaminants, fibronectin and IgM, were removed by affinity chromatography on gelatin- and anti-IgM-agarose, respectively. Finally, pure low-molecular-weight FVIII/vWF protein was harvested in the void volume fraction of a Sepharose CL-6B column. The smallest multimers had the size of the tetramer of the basic subunit chain of FVIII/vWF.     

Purified human factor VIII procoagulant protein: comparative hemostatic response after infusions into hemophilic and von Willebrand disease dogs.

The procoagulant protein F.VIII:C is noncovalently bound to von Willebrand factor (vWF) to give the factor VIII macromolecular complex. New highly purified preparations of isolated human F.VIII:C, devoid of vWF and about 500,000-fold purified, were administered to hemophilia A and von Willebrand disease (vWD) dogs to determine their hemostatic effectiveness and survival in the circulation. Two preparations of F.VIII:C were used: peak 1, with active components of Mr 185,000-280,000, and peak 2, with a single component of Mr 170,000. In hemophilic dogs, with no plasma F.VIII:C but normal vWF, both preparations immediately elevated plasma F.VIII:C to expected levels, promptly stopped induced and spontaneous hemorrhages, and gave sustained plasma levels of F.VIII:C. The isolated F.VIII:C immediately complexed with endogenous vWF in hemophilic plasma and was eliminated exponentially, with a half-life (t1/2) of about 9 hr. Survival of peak 2 F.VIII:C was longer than that of peak 1 material. In contrast, F.VIII:C complexed to vWF in a therapeutic concentrate administered to hemophilic dogs was eliminated biexponentially with first-phase t1/2 of 3.2 hr and second-phase t1/2 of 9 hr. In vWD dogs with no vWF and reduced F.VIII:C levels, the isolated F.VIII:C produced supernormal levels of F.VIII:C without effect on induced bleeding. It was rapidly eliminated from plasma with a t1/2 of about 1 hr, as was the complexed F.VIII:C in the concentrate. These data indicate that isolated F.VIII:C promptly complexes with vWF and in this form is highly effective in controlling hemophilic hemorrhages with good survival in plasma. Without endogenous vWF with which to complex, the F.VIII:C is promptly eliminated.     

Abnormal VIII: von Willebrand factor patterns in the plasma of patients with the hemolytic-uremic syndrome

Plasma VIII:von Willebrand factor antigen (VIII:vWF) levels were elevated approximately two- to eightfold in seven patients (three adults and four children) during acute episodes of thrombocytopenia, renal failure, and hemolytic anemia (the hemolytic-uremic syndrome, HUS). In all seven patients, there was an alteration in plasma VIII:vWF patterns during these acute HUS episodes, so that the largest VIII:vWF forms were relatively decreased. Plasma VIII:vWF multimer patterns returned to normal, or nearly to normal, as platelet counts returned to preexisting levels, even in the patients whose recovery of renal function was incomplete and whose plasma VIII:vWF antigen level remained above normal. The sister of one of the HUS patients had a similar clinical prodrome (gastroenteritis) that was not followed by thrombocytopenia or renal failure and was not accompanied by an elevated level or abnormal forms of plasma VIII:vWF. These results suggest that an alteration in VIII:vWF metabolism, distribution, or interaction with platelets is associated with acute HUS episodes. In contrast to patients with chronic relapsing thrombotic thrombocytopenic purpura, none of the HUS patients (either during or after the acute HUS episodes) had a defect in the conversion of unusually large VIII:vWF multimers derived from endothelial cells to the VIII:vWF forms found in normal plasma.     

不同年龄段大鼠血清影响内皮祖细胞向成熟内皮诱导分化

目的观察不同年龄段大鼠血清对骨髓内皮祖细胞向成熟内皮细胞诱导分化的影响。方法无菌取1~2月龄、19~26月龄Sprague-Dawley大鼠股骨和胫骨,获取骨髓单个核细胞,用含20%胎牛血清的DMEM/F12培养基培养,经差速贴壁法对二次贴壁细胞在纤维连接蛋白包被的培养板或培养瓶进行体外培养,以DiI-ac-LDL、FITC-UEA-1双荧光染色和FITC-CD31染色进行鉴定;收集制备相应年龄段(1~2月龄、19~26月龄)大鼠血清,将内皮祖细胞分为老年大鼠内皮祖细胞 老年大鼠血清组、老年大鼠内皮祖细胞 年轻大鼠血清组、年轻大鼠内皮祖细胞 老年大鼠血清组、年轻大鼠内皮祖细胞 年轻大鼠血清组。体外培养的内皮祖细胞经含20%不同年龄段大鼠血清的内皮条件培养基诱导培养后,激光共聚焦显微镜检测DiI-acLDLF、ITC-UEA-1双染色阳性率,免疫组织化学检测假血友病因子表达、逆转录聚合酶链反应分别检测内皮一氧化氮合酶、血管内皮生长因子受体2表达,体外血管生成实验观察内皮祖细胞参与管形生成能力。结果年轻大鼠血清显著提高老年大鼠内皮祖细胞在内皮条件培养基诱导培养后的DiI-acLDLF、ITC-UEA-1双染色阳性率(P<0.05),而老年大鼠血清显著降低年轻大鼠内皮祖细胞双染阳性率(P<0.05);年轻大鼠血清明显促进老年大鼠内皮祖细胞在内皮条件培养基诱导培养后的血管性血友病因子、内皮型一氧化氮合酶(P<0.01)及血管内皮生长因子受体2表达(P<0.05);年轻大鼠血清显著减少内皮条件培养基诱导培养后未参与血管生成的老年大鼠内皮祖细胞数(P<0.05)。结论年轻大鼠血清可显著增强老年大鼠内皮祖细胞向内皮细胞诱导分化的能力,而老年大鼠血清可部分抑制年轻大鼠内皮祖细胞向成熟内皮细胞诱导分化;年轻大鼠血清中可能存在激发衰老个体内皮祖细胞活力的系统性血清因子,这些因子可增强衰老个体来源的内皮祖细胞内在分化潜能。     

Involvement of low-density lipoprotein receptor-related protein (LRP) in the clearance of factor VIII in von Willebrand factor-deficient mice

Factor VIII is tightly noncovalently linked to von Willebrand factor (vWF) in plasma with a stoichiometry of 1:50, and vWF deficiency results in secondary factor VIII deficiency, with accelerated clearance of factor VIII from the circulation. We used a murine model of severe von Willebrand disease (vWF knockout mice) to study the effect of a recombinant vWF/pro-vWF preparation (rpvWF) on factor VIII survival and to investigate whether low-density lipoprotein receptor-related protein (LRP) might be involved in the in vivo clearance of factor VIII in the absence of vWF. vWF-deficient mice received 70 U/kg rpvWF in the first series of experiments, and in a second series, 80 mg/kg receptor-associated protein (RAP) as a recombinant fusion protein to block the action of LRP. Factor VIII levels were measured at time 0, or 1 or 3 hours after administration of rpvWF or RAP. RAP induced a sustained rise in factor VIII levels comparable to that induced by rpvWF. In a third series, the preadministration of RAP resulted in a slower disappearance of factor VIII antigen (measured by an enzyme-linked immunosorbent assay specific for human factor VIII) after infusion of recombinant factor VIII. These findings suggest that the accelerated clearance of factor VIII seen in the absence of vWF may be a result of the involvement of LRP in factor VIII metabolism. (Blood. 2000;95:1703-1708)     

Monovalent ionophores inhibit secretion of procollagen and fibronectin from cultured human fibroblasts

Procollagen and fibronectin are major products of confluent fibroblasts in culture and both are released from the cells. Procollagen is secreted by known pathways, while the mechanism of fibronectin release is controversial. We find that the secretion of both these proteins can be reduced to 20% by low concentrations (0.1-1 muM) of ionophores that have affinity for monovalent cations. In contrast, little effect upon secretion was found for similar concentrations of an ionophore that binds divalent cations. Electron microscopy showed that the inhibition of secretion is accompanied by accumulation of membranous vacuoles. We believe that the ionophores impede secretion by acting on the secretory structures rather than on the proteins themselves. Biochemical studies supported this interpretation because no changes were detected in hydroxylation or glycosylation of procollagen or glycosylation of fibronectin, nor were significant changes in cellular amino acid incorporation observed. Pulse-chase studies indicated that the rates of secretion were impaired by the ionophore without enhancing intracellular degradation. The decreased secretory rates accounted for the lower levels of procollagen and fibronectin in the culture medium; no evidence for increased catabolism of the secreted proteins was found. Secretion could be readily restored by removing the ionophore from the culture medium. The results indicate that procollagen and fibronectin may be simultaneously secreted, possibly utilizing a common pathway for secretion; the ionophores effectively interfere with cellular secretory pathways without impairing protein synthesis or protein glycosylation or altering protein catabolism.     

Inactivation of Hepatitis B and Hutchinson Strain Non-A,Non-B Hepatitis Viruses by Exposure to Tween 80 and Ether1

Titrated stocks of hepatitis B virus and Hutchinson strain non-A, non-B hepatitis virus were diluted in normal serum to contain, respectively, ≥10⁶ and ≥10⁴ chimpanzee infectious doses (CID₅₀) per milliliter and exposed to 1% Tween 80 and 20% ether at 4°C for 18 h. After evaporation of the ether, the treated sera were each inoculated into two chimpanzees. The animals remained free of serologic and biochemical evidence of hepatitis during a 6-month follow-up period, and were then shown to be susceptible to infection by challenge with the original untreated inocula. To assess the effect of exposure to Tween 80/ether on coagulation factors, four lots of antihemophilic factor (AHF) concentrate and 2 lots of commercial factor IX concentrate were treated as above. For the AHF concentrate there was an average of 70% recovery of factor VIII procoagulant activity, 93% recovery of factor VIII-related antigen, and 73% recovery of fibronectin opsonin activity and no detectable change in ristocetin cofactor activity or in fibronectin antigen. Crossed immunoelectrophoresis revealed no change in migration rate of fibrinogen, fibronectin, and von Willebrand factor (vWF), although the quantity of fibrinogen was reduced. Factor VIII procoagulant activity and vWF activity remained associated during chromatography on BioGel A15.     

The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid- phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.     

Reed-Sternberg cells in Hodgkin's disease contain fibronectin

Reed-Sternberg cells in the lymph nodes from five patients with Hodgkin's disease were studied. Indirect immunofluorescence on fixed sections with a monospecific anti-serum to fibronectin revealed abundant cytoplasmic fibronectin in approximately 90% of the Reed- Sternberg cells. In addition, the cells were shown by immunofluorescence to contain polyclonal IgG; however, factor VIII antigen, albumin, fibrinogen, alpha-2-macroglobulin, anti-thrombin III, and ceruloplasmin were not present. The abundant cytoplasmic fibronectin suggests that Reed-Sternberg cells are derived from tissue macrophages.     

Acquired von Willebrand Disease: Concise Review of Occurrence, Diagnosis, Pathogenesis, and Treatment

Acquired von Willebrand disease (AvWD) is a rare complication of an autoimmune or neoplastic disease. It is associated mostly with a lymphoid or plasma cell proliferative disorder. The clinical manifestations are similar to congenital von Willebrand disease. Diagnosis is confirmed by the demonstration of decreased levels of factor VIII coagulant activity (VIII:C), ristocetin cofactor activity (vWF:RCo), and von Willebrand factor (vWF) antigen (vWF:Ag). vWF multimer analysis usually reveals a type II defect with decreased abundance of higher molecular weight vWF multimers. Various pathogenetic mechanisms have been described, including the development of anti-vWF antibodies and adsorption of vWF by tumor cells. Successful management approaches have included treatment of the underlying disorder, infusion of high-dose gamma globulin, replacement therapy with factor VIII/vWF concentrates, intravenous infusion of desmopressin, and administration of corticosteroids.     

Rickettsia rickettsii infection of cultured endothelial cells induces release of large von Willebrand factor multimers from Weibel-Palade bodies.

The clinical manifestations of Rocky Mountain spotted fever (RMSF) result from Rickettsia rickettsii (R rickettsii) infection of endothelial cells and are mediated by pathologic changes localized to the vessel, including in situ thrombosis and tissue ischemia. This study uses in vitro infection of cultured human umbilical vein endothelial cells with R rickettsii to test the hypothesis that such infection induces von Willebrand factor (vWF) release from Weibel-Palade bodies, a process that could contribute to thrombotic changes. At 24 hours postinfection, there was an increase in metabolically prelabeled large multimers of vWF in the culture medium, with a concomitant decrease of these forms in the cell lysate samples. This release reaction was specific for the large multimer pool of vWF, localized to Weibel-Palade bodies, because no change in the distribution of dimeric forms between cells and culture medium was detected. Double-label immunofluorescence staining showed an inverse correlation between the number of R rickettsii and the number of Weibel-Palade bodies in infected cells. Cell lysis was minimal at 24 hours postinfection, as no detectable intracellular precursor forms (molecular weight 260,000) of vWF were released into the culture medium, there was no decrease in cell viability as measured by trypan blue exclusion, and no increase in 51Cr-release into the culture medium was observed when compared with uninfected controls. Release was likely a direct effect of the intracellular presence of the organism, rather than due to a noxious soluble factor such as endotoxin, because culture medium conditioned by infected endothelial cells was ineffective at inducing release in uninfected endothelial cell cultures. In summary, in vitro infection of endothelial cells by R rickettsii induces release of Weibel-Palade body contents, a process that may contribute to the pathogenesis of RMSF.     

Platelet-type von Willebrand's disease: characterization of a new bleeding disorder

An autosomally transmitted bleeding diathesis sharing some, but not all, features previously described in von Willebrand's disease (vWd) was studied in five patients representing three generations of a single family. Bleeding times in the upper normal range in conjunction with low-normal platelet counts, normal factor VIII coagulant activity and VIII-related antigen, decreased VIII-ristocetin cofactor activity, selective decrease of the higher molecule weight factor VIII/von Willebrand factor (VIII/vWF) multimers, and increased ristocetin- induced platelet agglutination at low ristocetin concentrations were characteristic. Binding of patient VIII/vWF to washed normal platelets was within normal limits, whereas binding of normal VIII/vWF to patient platelets was significantly increased (p less than 0.001 at 0.6 mg/ml ristocetin). This disorder accordingly appears to involve an intrinsic platelet abnormality affecting platelet-VIII/vWF interactions. It is proposed that the concept of vWD be broadened to include patients with this abnormality, which may appropriately be called "Platelet-type von Willebrand's disease."