No abstract

Summary. Factor (F)VIII functions as an enzymatic cofactor on the membranes of stimulated platelets. However, thrombin stimulates platelets to express only a small number of binding sites for FVIII. We wished to determine whether molecules that are likely to be present in a developing thrombus stimulate platelets to up-regulate FVIII binding site expression. Flow cytometry was utilized to measure binding of fluoresceinlabeled FVIIIa to activated platelets and a FXase assay was utilized to measure platelet-dependent function. Various agonists as well as normal and mutant fibrinogens and fibrin were evaluated as co-stimuli. Thrombin-stimulated platelets expressed 214 ± 67 binding sites for thrombin-activated FVIII (FVIIIa) and none of the established soluble agonists enhanced binding site exposure. However, the presence of 5 lg mL )1 fibrin increased the number of FVIIIa binding sites/platelet three-to eight-fold (1470 ± 130, range 600-1800) with a parallel increase in platelet-based FXase assay. Binding site upregulation was not stimulated by fibrinogen and was blocked by inhibitors of GPIIbIIIa. Mutant fibrin lacking the c-chain C-terminal four residues was ineffective while fibrin with altered RGD 2 sequences did stimulate expression of FVIIIa binding sites indicating that co-stimulation is mediated by the fibrin c-chain termini. Fibrin-enhanced expression of FVIIIa binding sites was not supported by D364H fibrin, which does not aggregate normally, and was blocked by the GPRP peptide, which inhibits fibrin polymerization. Polymerized fibrin can function as a platelet co-stimulus, up-regulating expression of binding sites for FVIIIa.

The rectal cuticle is permeable to H2PO4−, but much less so to HPO42−. Everted rectal sacs of Schistocerca gregaria transport PO4 from lumen to hemocoel side against large concentration and electrical differences. This active process is not caused by solvent drag and it obeys Michaelis–Menten kinetics. Entry of 32PO4 into rectal tissue from the lumen is inhibited by arsenate. Much of the 32PO4 is converted to organic forms in the tissue but these do not enter the hemocoel compartment. Net rates of PO4 movement across the rectal wall in vitro are high enough to explain recovery of phosphate secreted in situ by Malpighian tubules of starved locusts. The location and possible mechanism of PO4 transport are discussed.