Interchain disulfide bonds between the subunits in von Willebrand factor (vWf) dimers and in vWf multimers have been studied using some unique features of the cultured human umbilical vein endothelial cell system. Ammonium chloride inhibition of multimerization of vWf allowed selective examination of vWf dimeric molecules, and monoclonal antibody against the vWf propolypeptide was used to separate pro-vWf dimers from mature dimers. After cleavage of dimers and multimers with Staphylococcus aureus V-8 protease, the location of interchain disulfide bonds in amino (N)-terminal or carboxyl (C)-terminal fragments was determined by gel electrophoresis under reduced and nonreduced conditions. The first interchain disulfide bonds formed during dimerization are in the C-terminal region of the subunits, whereas interdimer disulfide bonds are located in the N-terminal portion. These data confirm recent electron microscopic projections of disulfide bond locations and provide support to the hypothetical role of the propolypeptide in the multimerization process.

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.

Heterogeneity in the human and rat plasma fibrinogen (FBG) gamma chains is due to differential splicing of the primary gamma chain mRNA transcript. The subunit composition of FBG containing the predominant form of the gamma chain is homodimeric (human: A alpha 2, B beta 2, gamma 50-gamma 50; rat: A alpha 2, B beta 2, gamma A-gamma A). The subunit composition of FBG containing the longer, minor form of the gamma chain is heterodimeric (human: A alpha 2, B beta 2, gamma 50- gamma 57.5; rat: A alpha 2, B beta 2, gamma A-gamma B). The variant gamma chain-FBGs comprise about 10% of the total plasma FBG in the human and 30% in the rat. Although the presence of homodimeric gamma B- gamma B FBG has been speculated, proof of its existence has been difficult to obtain experimentally. We now show that 5% to 6% of rat plasma FBG is found as homodimeric FBG with a subunit composition of A alpha 2, B beta 2, gamma B-gamma B. Commercially purified rat FBG was further purified by diethylaminoethyl-Sephacel chromatography with a combined pH and ionic strength gradient. The enriched gamma B-gamma B FBG fraction eluted at the lowest pH (6.3) and highest ionic strength (4.5 mmho) due to its increased net negative charge. To further purify gamma B-gamma B FBG, a Mono Q column with an NaCl gradient was used. The subunit composition of the purified gamma B-gamma B FBG was confirmed by its electrophoretic mobility under reducing and denaturing conditions both as FBG, and clotted and cross-linked fibrin. Homodimeric gamma B-gamma B FBG was unable to support adenosine diphosphate (ADP)-induced platelet aggregation, whereas heterodimeric gamma A-gamma B FBG was able to support ADP-induced platelet aggregation at 40% of that achieved with homodimeric gamma A-gamma A FBG, similar to previous observations with human FBGs. Taken together, these results support the hypothesis that the A alpha-RGD sites alone are not sufficient for dimeric FBG support of platelet aggregation. Furthermore, the data suggest that at least one intact platelet recognition site at the carboxyterminus of the gamma A or gamma 50 chain is required.

Increased neutrophil or HL60 cell adhesion to Rickettsia rickettsii- infected endothelial cells (ECs) was observed at 6 to 8 hours after the initiation of infection, diminishing by 24 hours. Similar increases were observed using formaldehyde-fixed neutrophils. Cellular association and likely the intracellular presence of rickettsiae was required for enhanced neutrophil adhesion, because culture medium conditioned by infected cells or rickettsiae rendered noninfective by pretreatment with tetracycline were ineffective at inducing neutrophil adhesion. Increases in neutrophil adhesion caused by infection were blocked by pretreatment of ECs with cycloheximide, suggesting the involvement of new protein synthesis in the cells' response. Flow cytometric analysis of infected cells showed increases in cell surface expression of E-selectin compared with uninfected control cells. Furthermore, incubation of 6- to 8-hour infected cells with a blocking monoclonal antibody against E-selectin (BB11) inhibited neutrophil adhesion an average of 61%. These results suggest the involvement of E- selectin in neutrophil adhesion to infected ECs occurring early in the course of the infection process. EC-initiated recruitment of neutrophil adhesion during rickettsiae infection could contribute to the pathologic changes associated with Rocky Mountain Spotted Fever.