# A minimum valency of 4 is required for robust activation of platelets in flow cytometry by multivalent nanobodies to Glycoprotein VI, C-type lectin-like receptor 2 and Platelet Endothelial Aggregation Receptor 1

## TL;DR

Tetravalent nanobodies to GPVI and CLEC-2 activate platelets more effectively than trivalent ones, helping improve flow cytometry assays for platelet function.

## Contribution

The study identifies that a minimum valency of 4 is needed for robust platelet activation by nanobodies targeting specific receptors.

## Key findings

- Tetravalent nanobodies to GPVI and CLEC-2 induce high platelet activation similar to collagen-related peptide.
- Trivalent nanobodies are partial agonists, showing less activation than tetravalent ones.
- Tetravalent PEAR1 nanobodies show variable activation across donors.

## Abstract

We have reported that trivalent and tetravalent nanobodies against glycoprotein (GP)VI, C-type lectin-like receptor (CLEC)-2, and platelet endothelial aggregation receptor (PEAR)1 stimulate powerful aggregation and adenosine triphosphate secretion in human platelets.

This study aimed to evaluate changes in platelet surface GPs elicited by activation of GPVI, CLEC-2, and PEAR1 using trivalent and tetravalent nanobodies.

The effect of the crosslinked nanobodies on P-selectin was measured in whole blood and washed platelets with and without secondary mediator inhibitors using classical flow cytometry and on 16 platelet surface GPs in whole blood using multispectral flow cytometry.

Trivalent nanobodies to GPVI and CLEC-2 stimulated modest (<60% of collagen-related peptide) expression of P-selectin in whole blood (10-fold dilution) and washed platelets (2 × 107 mL), whereas tetravalent nanobodies induced a response approaching that of collagen-related peptide. Stimulation of P-selectin expression was partially reduced by inhibitors of adenosine diphosphate (ADP) and thromboxane A2, indicating secondary platelet activation despite the low platelet concentration. By multispectral flow cytometry, tetravalent nanobodies to GPVI and CLEC-2 stimulated similar maximal fibrinogen binding and platelet surface α-granule (TLT-1 and CD154) and δ-granule (CD63) markers, but lower levels of the lysosomal marker CD107a. The tetravalent PEAR1 nanobody showed partial agonist activity in some donors but full activity in others.

Tetravalent nanobodies to GPVI and CLEC-2 stimulate powerful activation of platelets at low nanomolar concentrations in flow cytometry. In contrast, trivalent nanobodies are partial agonists. The defined stoichiometry of the nanobodies will aid development of standardized platelet flow cytometry assays.

•We previously showed that trivalent and tetravalent nanobodies to GPVI, CLEC-2 and PEAR1 stimulate powerful platelet aggregation and secretion as measured using LTA.•To investigate the minimum valency required for robust activation of each receptor we measured changes in multiple activation markers using classical and multispectral flow cytometry.•Tetravalent nanobodies to GPVI and CLEC-2, more than trivalent nanobodies, induced consistent, high, platelet surface expression of α- δ- and lysosomal granule markers and corresponding populations of highly activated platelets across donors.•Activation with tetravalent PEAR1 nanobodies was less robust, varied between donors, and produced fewer highly activated platelets.•The defined stoichiometry of the nanobodies will aid development of standardised platelet flow cytometry assays which may be useful for assessing patients with platelet function disorders.

We previously showed that trivalent and tetravalent nanobodies to GPVI, CLEC-2 and PEAR1 stimulate powerful platelet aggregation and secretion as measured using LTA.

To investigate the minimum valency required for robust activation of each receptor we measured changes in multiple activation markers using classical and multispectral flow cytometry.

Tetravalent nanobodies to GPVI and CLEC-2, more than trivalent nanobodies, induced consistent, high, platelet surface expression of α- δ- and lysosomal granule markers and corresponding populations of highly activated platelets across donors.

Activation with tetravalent PEAR1 nanobodies was less robust, varied between donors, and produced fewer highly activated platelets.

The defined stoichiometry of the nanobodies will aid development of standardised platelet flow cytometry assays which may be useful for assessing patients with platelet function disorders.