Abstract
Introduction: Plasma fibronectin (FN) is synthesized by hepatocytes and secreted into the circulation in a soluble, compact and non-fibrillar form. Plasma FN is assembled by cells or adherent platelets into functional fibrils. Reports have indicated that the process to incorporate FN into multimer fibrils can also occur in cell-free models in vitro by incubation with denaturants, reducing agents, or anastellin (FN peptidic fragment). Here, we report on (1) the formation of insoluble fibrillar-like supramolecules of plasma FN (FN fibrils) by exposing the molecules to increasing shear rates and (2) the functional characterization in platelet adhesion and aggregation.
Methods: To induce the formation of FN fibrils, 1 ml of plasma FN solution (100 μg/ml) was added to plates pre-coated with FN (100 μg/ml). Subsequently, the FN solutions were exposed to shear (50 to 5000 s-1 within 5 min and subsequently 5000 to 50 s-1 within 5 min) generated by a cone-plate rheometer (Haaka Rheostress 1; Thermo Scientific). Viscosities of FN solutions were recorded. To quantify the formation of FN fibrils, FN solutions after exposure to shear were collected and incubated with 2% deoxycholate (DOC). The DOC-insoluble pellets containing FN fibrils were isolated by centrifugation at 19,000 g for 20 min at 4°C and resuspended in 1% SDS buffer for Western blot analyses. For adhesion experiments, washed platelets (107/ml) in HEPES Tyrode’s buffer were labeled with 10 μM 5-chloromethylfluorescein diacetate and placed on 96-well plates pre-coated with FN or FN fibrils (25 µg/ml) for 30 min at 37°C. In parallel experiments, platelets resuspended in FN-depleted plasma (107/ml) were placed onto immobilized collagen, fibrinogen, FN (10 µg/ml) in the presence of FN (300 µg/ml) or FN fibrils (10 µg/ml). For aggregation experiments, FN (5, 10, 300 µg/ml) or FN fibrils (5, 10 µg/ml) was added to platelet-rich plasma (PRP) or platelets resuspended in FN-depleted plasma (2.5 × 108/ml). Aggregation was induced by 400 nM PMA (Phorbol 12-myristate 13-acetate), or 10 µg/ml collagen.
Results: The initial viscosities (mPa's) of FN solutions were 7.62 ± 0.98. Upon exposure to dynamic shear for 10 min, viscosities increased to 10.98 ± 1.81 (p = 0.02, n = 4), suggesting conformational changes of FN. Western blot analyses of DOC-insoluble fractions revealed bands of FN in the range of 220 – 250 kDa (reducing condition), indicative of the formation of insoluble fibrils in FN solutions after exposure to shear. Platelet adhesion and aggregation experiments were performed to compare the activity of FN fibrils with normal plasma FN. For adhesion experiments, washed platelets in HEPES Tyrode’s buffer were placed onto immobilized FN or FN fibrils (25 µg/ml). The adhesion rates (mean fluorescence signal ± SD) of washed platelets were higher onto surfaces coated with FN fibrils (0.5 ± 0.06) than onto surfaces coated with plasma FN (0.4 ± 0.01) (p = 0.04, n = 3). In parallel adhesion experiments using platelets resuspended in FN-depleted plasma, addition of plasma FN (300 µg/ml) increased platelet adhesion rates onto immobilized collagen (from 0.14 ± 0.005 to 0.2 ± 0.01, p = 0.0007), fibrinogen (from 0.16 ± 0.03 to 0.22 ± 0.01, p = 0.03), and FN (from 0.14 ± 0.01 to 0.18 ± 0.02, p = 0.04) (n = 3). Addition of FN fibrils at low concentration of 10 µg/ml had a similar supportive effect. FN showed an inhibitory effect in platelet aggregation. Activation by 400 nM PMA induced aggregation of PRP by 81% (amplitude). In the presence of plasma FN at 5, 10, 300 µg/ml, platelet aggregation was reduced to 50 %, 41 %, and 29.5 %, respectively. A stronger inhibition on platelet aggregation was seen when FN fibrils were added. PRP aggregated by 35.4 % and 17 % in the presence of 5 and 10 µg/ml FN fibrils, respectively. The same phenomenon was observed in aggregation assays using platelets resuspended in FN-depleted plasma and collagen (10 µg/ml) as activating agonist.
Conclusion: Our study shows that dynamic shear rates induce the formation of insoluble fibrillar-like form of plasma FN in cell-free model in vitro. Fibril formation of FN can be monitored by measuring viscosities of FN solutions during exposure to shear and quantified by Western blot. Shear-induced formed FN fibrils have an explicitly stronger activity in supporting platelet adhesion and inhibiting platelet aggregation than normal plasma FN. This finding emphasizes the importance of FN assembly on its activity in platelet functions.
Disclosures
No relevant conflicts of interest to declare.
Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin- dependent platelet adhesion under flow conditions
