BFPI: A Minimal Prothrombinase Inhibitor from the Saliva of Simulium Vittatum

Background: The black fly, Simulium Vittatum, has an anticoagulant protein in its saliva that allows it to feed on mammalian blood (black fly protease inhibitor; BFPI). Remarkably, BFPI is similar to the human anticoagulant tissue factor pathway inhibitor alpha (TFPIα). TFPIα contains three Kunitz-type protease inhibitor domains (K1, K2, K3), which inhibit factor VIIa (FVIIa) and factor Xa (FXa) and bind the co-factor protein S (PS), respectively; BFPI contains a single Kunitz domain that inhibits FXa. In addition, TFPIα and BFPI contain homologous basic regions (BRs) near their C-termini (252LIKTKRKRKK261 in human TFPIα, LIKTRKRKPKK in BFPI). The TFPIα BR binds a regulatory acidic region (AR) in factor Va (FVa). The AR is present in forms of FVa released by collagen-activated platelets and generated through limited proteolysis by FXa (FVaXa), and is removed by thrombin (FVaIIa). We hypothesized that BFPI, through its Kunitz domain and basic C-terminus, inhibits early forms of the prothrombinase complex, but does not possess the other inhibitory functions of TFPIα: (1) K1-dependent inhibition of the tissue factor (TF)-FVIIa complex; and (2) PS/K3-dependent FXa inhibition. Results: Recombinant BFPI inhibited FXa in an amidolytic activity assay, and PS did not promote this inhibition. BFPI did not inhibit TF-FVIIa-mediated FX activation. As described with TFPIα, FV promoted FXa inhibition by BFPI but FVaIIa did not, suggesting that the BFPI BR is capable of binding the FVa AR. In a purified protein assay, BFPI inhibited prothrombinase assembled with FVaXa (IC50=4.9nM), but not FVaIIa. Similarly, 5nM BFPI increased the lag time for FXa-initiated plasma thrombin generation by 10.4±1.5%. We next used BFPI as a backbone to evaluate a reported human mutation in the TFPIα BR, K254E. Every mammalian, avian, or reptilian TFPIα sequence available contains either a Lys or Arg residue at this position, suggesting that this residue is functionally important. In purified protein assays, BFPI-K254E inhibited FXa amidolytic activity identically to BFPI, but FV did not promote this inhibition, suggesting that BFPI-K254E has a specific defect in its interaction with FV. Consistent with this, BFPI-K254E was a weaker inhibitor of prothrombinase assembled with FVaXa (IC50 = 15.8nM) and FXa-initiated plasma thrombin generation. The results obtained with BFPI-K254E were confirmed using peptides and full-length TFPIα proteins. First, a peptide mimicking the wild type TFPIα BR (LIKTKRKRKK) inhibited prothrombinase assembled with FVaXa (IC50 = 1.0 µM), while the substituted peptide (LIETKRKRKK) was substantially weaker (20% inhibition observed with 340 µM peptide). Second, full-length TFPIα-K254E was a weaker inhibitor of prothrombinase containing FXa-activated FVa (IC50 = 14.8 nM, vs. 1.8 nM for TFPIα) and had greatly reduced anticoagulant activity in plasma-based thrombin generation assays. Conclusions: In summary, the anticoagulant effect of BFPI is mediated through inhibition of early forms of prothrombinase, independent of TF-FVIIa inhibition or PS-dependent FXa inhibition. The natural mutation TFPIα K254E disrupts prothrombinase inhibition, despite the presence of six other conserved basic residues, and is thus procoagulant in human plasma. The absolute conservation of the TFPIα BR, and its usurpation to allow feeding by black flies, point to formation of the initial prothrombinase complex as a key regulatory step in blood coagulation. Disclosures Mast: Novo Nordisk: Research Funding.

Pathogenesis of Vesicular Stomatitis New Jersey Virus Infection in Deer Mice (Peromyscus maniculatus) Transmitted by Black Flies (Simulium vittatum)

The natural transmission of vesicular stomatitis New Jersey virus (VSNJV), an arthropod-borne virus, is not completely understood. Rodents may have a role as reservoir or amplifying hosts. In this study, juvenile and nestling deer mice ( Peromyscus maniculatus) were exposed to VSNJV-infected black fly ( Simulium vittatum) bites followed by a second exposure to naive black flies on the nestling mice. Severe neurological signs were observed in some juvenile mice by 6 to 8 days postinoculation (DPI); viremia was not detected in 25 juvenile deer mice following exposure to VSNJV-infected fly bites. Both juvenile and nestling mice had lesions and viral antigen in the central nervous system (CNS); in juveniles, their distribution suggested that the sensory pathway was the most likely route to the CNS. In contrast, a hematogenous route was probably involved in nestling mice, since all of these mice developed viremia and had widespread antigen distribution in the CNS and other tissues on 2 DPI. VSNJV was recovered from naive flies that fed on viremic nestling mice. This is the first report of viremia in a potential natural host following infection with VSNJV via insect bite and conversely of an insect becoming infected with VSNJV by feeding on a viremic host. These results, along with histopathology and immunohistochemistry, show that nestling mice have widespread dissemination of VSNJV following VSNJV-infected black fly bite and are a potential reservoir or amplifying host for VSNJV.

Drift and activity responses of black flies (Simulium vittatum) in the field: influences of tactile and injury-released stimuli from simulated predation

Prey must manage threat from many types of predators; therefore, selection should favor sensory mechanisms that allow the refinement of defensive behavior. To assess responses to tactile and chemical stimuli related to predation, we observed drift and activity of larval black flies (Simulium vittatum) to simulated predator contact intended to imitate benthic and drift predators as well as a combination of tactile and injury-released stimuli. In the field, larvae responded to tactile stimuli applied to the head with a higher frequency of curling and posterior abdominal segments with a higher frequency of drifting. Additionally, chemical cues from injured conspecifics followed by tactile stimuli applied to the head resulted in a higher frequency of drifting than to either cue independently and this effect was more pronounced at night. The results of our study indicate that larval black flies may utilize multiple cues to determine their antipredator and predator avoidance strategies.