Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes.

Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ∼55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ∼5,000- and ∼80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ∼85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.

Plasminogen substrate recognition by the streptokinase-plasminogen catalytic complex is facilitated by Arg253, Lys256, and Lys257 in the streptokinase beta-domain and kringle 5 of the substrate.

Streptokinase (SK) conformationally activates the central zymogen of the fibrinolytic system, plasminogen (Pg). The SK.Pg* catalytic complex binds Pg as a specific substrate and cleaves it into plasmin (Pm), which binds SK to form the SK.Pm complex that propagates Pm generation. Catalytic complex formation is dependent on lysine-binding site (LBS) interactions between a Pg/Pm kringle and the SK COOH-terminal Lys(414). Pg substrate recognition is also LBS-dependent, but the kringle and SK structural element(s) responsible have not been identified. SK mutants lacking Lys(414) with Ala substitutions of charged residues in the SK beta-domain 250-loop were evaluated in kinetic studies that resolved conformational and proteolytic Pg activation. Activation of [Lys]Pg and mini-Pg (containing only kringle 5 of Pg) by SK with Ala substitutions of Arg(253), Lys(256), and Lys(257) showed decreases in the bimolecular rate constant for Pm generation, with nearly total inhibition for the SK Lys(256)/Lys(257) double mutant. Binding of bovine Pg (BPg) to the SK.Pm complex containing fluorescently labeled Pm demonstrated LBS-dependent assembly of a SK.labeled Pm.BPg ternary complex, whereas BPg did not bind to the complex containing the SK Lys(256)/Lys(257) mutant. BPg was activated by SK.Pm with a K(m) indistinguishable from the K(D) for BPg binding to form the ternary complex, whereas the SK Lys(256)/Lys(257) mutant did not support BPg activation. We conclude that SK residues Arg(253), Lys(256), and Lys(257) mediate Pg substrate recognition through kringle 5 of the [Lys]Pg and mini-Pg substrates. A molecular model of the SK.kringle 5 complex identifies the putative interactions involved in LBS-dependent Pg substrate recognition.