PB0889 - Biodesigned recombinant protein mimicking activities of fibrillar collagen in primary haemostasis

Background: Collagens play an important role in primary haemostasis. Most of reagents use fibrillar collagen extracted from natural tissues, with difficulties for batch-to-batch reproducibility. Up to now a few peptides have been obtained by chemical synthesis and thoroughly studied, mostly regarding platelet activation via GPVI, such as collagen-related-protein (CRP).

Aims: We report scale-up synthesis and characterization of a short non-fibrillar recombinant protein and demonstrate that important functional properties of fibrillar/native collagen in primary haemostasis are well mimicked.

Methods: The biodesigned triple-helical protein NVH020, of apparent MM of 55 kDa, containing the binding motifs to VWF, α2β1, and GPVI, was produced in a genetically modified yeast. It does not structure itself as a fibrillar form but as a particulate one (atomic-force microscopy). Soluble in water pH 7.4, it is easy to handle

Results: Two batches were thoroughly studied, in comparison with collagen Horm (Takeda). Binding capacity to VWF, α2β1, and GPVI was demonstrated in solid-phase immunoassays. Platelets in suspension (citrated PRP – ten healthy subjects) were activated in closed systems (NVH020 at 2.5-10 μg/mL): (i) light transmission aggregometry and lumiaggregometry (ATP release) with tracings close to those obtained with Horm collagen, including the typical lag-phase; (ii) flow cytometry with induction of PAC-1 binding (activated αIIbβ3), CD62 P-selectin exposure and annexin-V binding (phosphatidylserine exposure).
Importantly, flow experiments (Cellix chambers; shear rate 1,500/s) with PPACK-anticoagulated whole blood showed the ability of NVH020 to make platelet adhere and accumulate. A more uniform and reproducible coating of the protein than with fibrillar collagen is expected.

Conclusion(s): This small size triple-helical protein NVH020 mimics the properties of natural fibrillar collagen in primary haemostasis and is a promising candidate as a research and diagnostic tool for laboratory experiments and tests respectively.