The basement membrane protein nidogen-1 supports platelet adhesion and activation

Abstract

The core structure of the extracellular basement membrane is made up of self-assembling networks of collagen and laminin which associate with each other through the bridging adapter proteins including the sulfated monomeric glycoprotein nidogen. While collagen and laminin are known to support platelet adhesion and activation via β1 integrins and glycoprotein (GP) VI, respectively, whether nidogen contributes to platelet activation and hemostasis is unknown. In this study, we demonstrate that recombinant human nidogen-1 supports platelet adhesion and stimulates platelet activation in a phospholipase-C γ-2 (PLCγ2), Src and Syk kinase-dependent manner downstream. Platetet adhesion to nidogen-1 was inhibited by blocking the platelet receptors GPVI and β1 integrins. Platelet adhesion to nidogen-1 activated the IκB kinase (IKK) complex, while pharmacological inhibition of IKK blocked platelet spreading on nidogen. Taken together our results suggest that nidogen may play a redundant role in hemostasis by activating platelets downstream of GPVI.

Keywords: Extracellular matrix proteins; hemostasis; nidogen; platelet.

Figure 1.. Adhesion and spreading of human platelets on nidogen-1.

A. Human washed platelets (2 × 10⁷/mL) were placed on coverslips coated with BSA (5 mg/mL), fibrinogen (100 μg/mL), soluble collagen (50 μg/mL), or recombinant human nidogen-1 (100 μg/mL) for 45 min at 37° C and imaged using differential interference contrast (DIC) microscopy. Images are representative of 3 independent experiments. Scale bar, 10 μm. B. The number of adherent platelets on BSA, fibrinogen (FG), soluble collagen (CL) and nidogen-1 (ND) for increasing platelet concentrations and increasing protein concentrations were recorded for 3 fields of view and expressed as mean ± SEM from at least 3 different experiments. C. Lysates from washed human platelets seeded on coverslips coated with recombinant nidogen-1, fibrinogen, CRP-XL (collagen related peptide), fibrillar collagen, or quiescent platelets (resting) in solution were analyzed for total phosphoprotein content with 4G10 or phosphorylation of Syk pY525, BTK pY223 (Bruton’s tyrosine kinase), BTK pY551, activation of PKC (protein kinase C), PLCγ2 pY1217 (phospholipase Cγ2), or activation of NF-κB through the analysis of IKK and p65 phosphorylation by western blotting (WB). Protein molecular markers on the right.

Figure 2.. Effect of platelet inhibitors on platelet adhesion and spreading on nidogen-1.

Human washed platelets (2 × 10⁷/mL) were pre-treated with A. vehicle (DMSO), Syk inhibitor (Bay-61–3606, 5 μM), SFK inhibitor (PP2, 10 μM), PI3K inhibitor (wortmannin, 100 nM), C. PLC inhibitor (U73122, 10 μM), PLC inhibitor analog (U73343,10 μM) or IKK inhibitor (IKK-16, 10 μM) E. vehicle (DMSO) or anti-GPVI (ACT017 GPVI inhibitor, 40 μg/mL) H. vehicle (DMSO), anti- β₁ (AIIB2, 20 μg/mL), anti-αIIbβ3 (integrillin, 20 μg/mL) (data not shown) or ADP scavenger (apyrase, 2U/ml) for 15 min prior to seeding on coverslips coated with recombinant nidogen-1 (100 μg/mL) for 45 min at 37° C and imaged using differential interference contrast (DIC) microscopy. Images are representative of at least 3 independent experiments. B and D. Number of adherent platelets and mean surface area of platelet on nidogen-1 were recorded for 3 fields of view for each condition and expressed as mean ± SEM. F and H. Number of adherent platelets was recorded for 3 fields of view for each condition and expressed as mean ± SEM. * P < 0.05 with respect to platelet adhesion in the absence of inhibitors. Scale bar, 10 μm.