Activation peptides prolong the murine plasma half-life of human factor VII

Coagulation factors VII (FVII), IX (FIX), X (FX), and protein C share the same domain organization but display very different plasma half-lives. It is plausible that the half-life is influenced by the activation peptide, differing in length and glycosylation and missing in FVII. To test this hypothesis, the influence of activation peptides on the plasma half-life of human FVII was studied by administering human FVII variants containing activation peptide motifs to mice. Insertion of the activation peptide from FX gave 4-fold longer terminal half-life (5.5 hours vs 1.4 hours for FVII), whereas the activation peptide from FIX and protein C resulted in half-lives of 4.3 and 1.7 hours, respectively. Using FX's activation peptide we identified the N-linked glycans as structural features important for the half-life. The peptide location within the FVII molecule appeared not to be critical because similar prolongation was obtained with the activation peptide inserted immediately before the normal site of activation and at the C-terminus. However, only the latter variant was activatable, yielding full amidolytic activity and reduced proteolytic activity with preserved long half-life. Our data support that activation peptides function as plasma retention signals and constitute a new manner to extend the half-life of FVII(a).

Heteromeric and Homomeric Transmembrane Domain Associations Regulate αIIbβ3 Function.

The integrin αIIbβ3 resides on the platelet surface in an equilibrium between inactive and active conformations that can be shifted in either direction by altering the distance between the stalks that anchor αIIbβ3 in the platelet membrane. Accordingly, the αIIb and β3 transmembrane (TM) domains, located near the ends of the stalks, are in proximity when αIIbβ3 is inactive and separate upon αIIbβ3 activation. Peptides corresponding to these domains undergo both homomeric and heteromeric interactions in biological membranes. Thus, it is possible that the shift between inactive and active αIIbβ3 conformations is accompanied by a shift from heteromeric to homomeric αIIb and β3 TM domain interactions. Indeed, we reported that introducing Asn, a residue known to strengthen homomeric TM helix interactions, into the β3 TM domain shifts αIIbβ3 to an active state. As a further test of this model of αIIbβ3 regulation, we studied the effects of mutations of the αIIb TM domain. First, we placed Asn at 10 consecutive positions in the αIIb TM domain, extending from residues V969 to L978, and co-expressed each mutant with WT β3 in CHO cells. Only one of the mutants, G972N, was constitutively active, binding ~ 8-fold more fibrinogen than WT αIIbβ3. Moreover, G972N was expressed in non-uniform patches on the CHO cell surface, consistent with the formation of αIIbβ3 clusters. G972 is the first residue of a GxxxG motif that is essential for dimerization of the αIIb TM domain. Using the TOXCAT assay to assess TM domain dimerization, we observed that G972N results in a 55% decrease in TOXCAT activity. This implies that the effect of G972N on the αIIbβ3 activation state was not a result of increased homo-dimerization of αIIb, but it is more likely that the mutation disrupted its heteromeric interaction with β3. To test this suggestion, we introduced mutations known to disrupt αIIb homo-dimerization (G972L, G976A, and G976L) into αIIbβ3 and measured their effect on αIIbβ3 function. Like G972N, each mutation induced constitutive αIIbβ3 activation and clustering. Lastly, we measured the effect of L980A, a mutation in the αIIb TM domain that unlike G972N, results in a 2.5-fold increase in TOXCAT activity. CHO cells expressing L980A constitutively bound ~ 6.5-fold more fibrinogen than did cells expressing WT αIIbβ3. Taken together, our results suggest a mechanism for αIIbβ3 regulation that involves both the heteromeric and homomeric association of the αIIb and β3 TM domains. Any process that destabilizes the heteromeric association of the αIIb and β3 TM domains would be expected to allow dissociation of these domains with concomitant αIIbβ3 activation. Hence, mutations that disrupt the heteromeric αIIb/β3 TM domain interface “push” αIIbβ3 toward activation. Conversely, intermolecular interactions that either require separation of the αIIb and β3 TM domains or are more favorable when they dissociate, such as homo-oligomerization of the αIIb and β3 TM domains, will “pull” the equilibrium toward the activated state.

Effect of prothrombin and its activation fragments on calcium oxalate crystal growth and aggregation in undiluted human urine in vitro: relationship between protein structure and inhibitory activity

In recent years there has been great interest in the putative role of prothrombin and its activation peptides, especially the urinary form of prothrombin fragment 1, in the pathogenesis of calcium oxalate (CaOx) urolithiasis. Previously, we showed that prothrombin and its activation peptides inhibit CaOx crystallization in inorganic conditions in vitro. The aim of the present study was to determine if this inhibitory activity is retained in undiluted human urine and, therefore, whether it is likely to have any influence under physiological conditions. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. Prothrombin was purified from Prothrombinex-HT, cleaved with thrombin and the resulting fragment 1 (F1) and fragment 2 (F2) were purified. The purity of each protein was confirmed by SDS/PAGE, and their effects on CaOx crystallization in undiluted ultrafiltered human urine were determined at a final concentration 80.65nmol/l using Coulter Counter and [14C]oxalate analysis. The precipitated crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that, whereas prothrombin and its activation peptides did not affect the urinary metastable limit and the size of the precipitated particles, F1 did significantly reduce the latter. These findings were corroborated with scanning electron microscopy which also revealed that the reduction in particle size caused by F1 resulted from a decrease in the degree of crystal aggregation, rather than in the size of the individual crystals. The [14C]oxalate data showed that none of the proteins added significantly inhibited the mineral deposition. It was concluded that with the exception of F1, which does inhibit CaOx crystal aggregation, prothrombin and its activation peptides do not alter the deposition and aggregation of CaOx crystals in ultrafiltered human urine in vitro. Also, the γ-carboxyglutamic acid domain of prothrombin and F1, which is absent from thrombin and F2, is the region of the molecules that determines their potent inhibitory effects. The superior potency of F1, compared with prothrombin, probably results from the molecule's greater charge-to-mass ratio.