We have previously demonstrated that increasing factor XIII concentrations above that present in plasma (1 U/mL) results in the formation of very high molecular weight alpha fate polyacrylamide and agarose gel electrophoresis (SDS-PAGE). In this report, we have examined the effect of such crosslinking on plasmic susceptibility of fibrin prepared from purified fibrinogen and from plasma in the presence of factor XIII concentrations between 0 and 10 U/mL. The crosslinking achieved with purified fibrinogen at 1 U/mL factor XIII increased resistance to plasmic degradation by 32% as measured in a radiolabeled clot lysis system. However, further increases in plasmic resistance occurred at factor XIII concentrations of 2 and 10 U/mL, the latter decreasing the lysis rate to 45% of that which occurred in the absence of factor XIII. To achieve the same rate of clot lysis with fibrin formed using 10 U/mL rather than 1 U/mL of factor XIII, an increase in plasmin concentration of up to 4.2-fold was required. Similar results were obtained using clots prepared from plasma in the presence of factor XIII concentrations greater than 1 U/mL. Since the alpha 2-plasmin inhibitor content was the same for fibrin at 1 or 10 U/mL factor XIII, the increasing plasmic resistance could not be attributed to increased binding of the inhibitor. We conclude that fibrin prepared in the presence of factor XIII at concentrations exceeding that in plasma shows increased resistance to plasmic degradation, which is likely explained by the formation of very high molecular weight alpha polymer chains.
Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies (WPB) of cultured endothelial cells following treatment with a secretagogue, whereas predominantly dimeric forms are secreted constitutively. These two pools of vWf were used to compare binding of the various multimeric forms of vWf to the extracellular matrix (ECM), the in vitro model of the basement membrane. The released multimers and an equal number of subunits of constitutively secreted vWf were placed, for 72 hours, on cultures of human foreskin fibroblasts (HFF) grown on glass coverslips, then fixed and stained by fluorescence using anti-vWf antiserum. Constitutively secreted vWf produced only a trace of matrix decoration, whereas the released large multimers bound more extensively. In order to determine if increased binding of released vWf was due to the presence of another component in the releasate, releasate from which vWf was adsorbed was combined with constitutively secreted vWf, and this mixture was overlaid onto HFF. The presence of the adsorbed releasate did not promote binding of constitutively secreted vWf. Therefore, it appears that the enhanced binding observed was due to the large multimeric size of vWf stored in the WPB. To further substantiate this, iodinated plasma vWf which was presumably constitutively secreted from endothelial cells was overlaid on.to HFF for 72 hours, labeled vWf was removed, and cells were washed extensively and lysed. Samples of iodinated plasma vWf (starting material) and cell lysates were e1ectrophoresed, non-reduced on an agarose gel. Densitometric scans of starting material and of bound vWf revealed that the large multimeric forms bound preferentially. It appears that multivalency is likely an important property in vWf interaction with the ECM, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the WPBs, therefore, is not only especially suited for platelet interaction, but also for interaction with the ECM.
Thrombin cleaves fibrinopeptides from fibrinogen, converting it to fibrin monomer, and activates factor XIII, which catalyzes the formation of intermolecular c-(y-glutamyl)-lysine bonds to stabilize the fibrin polymer. The formation of factor XIIla-catalyzed fibrin polymers during clotting of plasma and purified fibrinogen in vitro was followed by a sodium dodecyl sulfate agarose gel technique, and an increase in both amount and size of y-chain cross-linked polymers was demonstrated before visible clot formation. Plasma from patients presenting with acute myocardial infarction showed increases in the plasma concentration of fibrin polymer and in the proportion of total fibrinogen present as polymer, as determined by a quantitative adaptation of the electrophoretic technique. The plasma concentration in patients with subendocardial or transmural myocardial infarction showed significant (p < .005) increases to 4.0 1.0% and 3.6 + .8%, respectively, as compared with the concentration in normal plasma (0.8 + 0.1 %). There was no difference in plasma concentration in samples from patients with transmural compared with those with subendocardial myocardial infarction. This study provides the first demonstration of factor XIIIa cross-linked fibrin polymers in thrombotic disease and indicates the presence of increased activity of both thrombin and factor XIIIa in patients with acute myocardial infarction.
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.
Microvascular thrombi underlie many of the clinical manifestations of Rocky Mountain spotted fever (RMSF), a disease characterized by Rickettsia rickettsii infection of vascular endothelial cells. Studies were designed to determine whether R rickettsii-infection of cultured human umbilical vein endothelial cells results in tissue factor (TF) induction, a process that could directly activate coagulation in infected vessels. Whereas uninfected endothelial cell cultures showed essentially undetectable TF mRNA and activity, both TF mRNA and activity were present after R rickettsii infection. TF mRNA levels were transient, peaking at 4 hours after the initiation of infection, whereas the peak of TF activity occurred at 8 hours. Induction of the TF response requires the intracellular presence of R rickettsii organisms, because uninfected rickettsia were ineffective and the response was blocked by inhibiting rickettsial entry using cytochalasin B. TF induction was not mediated by endothelial cell release of soluble factor, because no response was induced using culture medium conditioned by R rickettsii-infected cells. Furthermore, preadsorption of suspensions of R rickettsii with polymyxin B to remove contaminating lipopolysaccharide did not eliminate the TF response. Induction of TF in vital endothelial cells during R rickettsii infection could be the trigger for vascular thrombus formation of RMSF.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.