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.

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