A s bstract. We have examined the role of heparinlike molecules in the regulation of coagulation by perfusing rat hindquarters with purified human thrombin and with its plasma inhibitor, antithrombin. Our data indicate that contact of the hemostatic components with the endothelium enhances the rate of thrombin-antithrombin complex formation by as much as 19-fold over the uncatalyzed rate of enzyme-inhibitor interaction. Heparinlike molecules are responsible for the antithrombin accelerating activity. The amount of thrombin-antithrombin complex generated within the hindlimb preparation after pretreatment of the vasculature with purified Flavobacterium heparinase or with addition of platelet Factor IV to the hemostatic components, was equal to the uncatalyzed levels. These heparinlike molecules appear to be tightly bound to the luminal surface of the endothelium, since they could not be detected within the physiologic buffer that was perfused through the animal. The above mucopolysaccharides function in a manner similar to commercial heparin, since modification of antithrombin at a site critical for heparin-dependent acceleration of the protease inhibitor resulted in a level of interaction product identical to the uncatalyzed amount. Finally, addition of diisofluorophosphate-thrombin to the enzyme perfusion stream reduced the amount of thrombin-antithrombin complex formed in the animal by 30-40%, which suggested that thrombin bound to the en-

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To assess the contribution of mast cells to the maintenance of blood fluidity, the hindlimb vasculature of mast cell-deficient mice (W/Wv) and littermates containing normal levels of mast cells (+/+), were perfused with purified human thrombin and antithrombin. Enzyme-inhibitor complex generation within the vasculature was enhanced to a comparable extent for W/Wv and +/+ mice over the uncatalyzed rate, that level of complex produced within a similar time interval in the absence of heparin. Perfusion of purified Flavobacterium heparinase prior to infusion of the hemostatic components, or perfusion of antithrombin modified at the heparin-binding domain, reduced W/Wv and +/+ hindlimb thrombin-antithrombin complex formation to the uncatalyzed rate. To further define the cellular source of the vascular-associated heparin-like molecules, endothelial cells isolated from epididymal fat pads of W/Wv and +/+ mice were grown in vitro. The acceleration of thrombin-antithrombin interactions in the presence of endothelial cell-derived glycosaminoglycans was similar for W/Wv and +/+ mice, was abolished with purified bacterial heparinase, and was expressed to only a minor extent when utilizing modified antithrombin. The biologically active mucopolysaccharides appear to be present on the cell surface.

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In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.