Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces

Abstract

Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin α₂β₁. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca²⁺. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)_n and Horm-type collagen evoked Syk-dependent Ca²⁺ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)_n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)_n sequence.

Keywords: calcium; collagen; glycoprotein VI; platelet activation; protein tyrosine kinase; thrombus.

Figure 1

Syk inhibition affecting platelet Ca²⁺ rises by collagen peptides with (GPO)_n motif. Fura-2-loaded platelets in 96-well plates were pre-incubated with Syk-IN (5 µM) or left untreated before stimulation with collagen peptide (M1-5, 10 µg/mL). Changes in [Ca²⁺]_i were recorded over time per well-plate row by ratio fluorometry using a FlexStation 3. Peptides were injected into wells at 60 s (arrow) and reached platelets in a diffusion-limited way. (A) Calibrated [Ca²⁺]_i traces recorded over 600 s in the absence (black, control) or presence (gray) of a Syk inhibitor. Traces are representative of three experiments. (B) Quantification for M1-5 of increased [Ca²⁺]_i at 300 s (top graph) or 600 s (bottom graph). Means ± SEM (n = 3). Paired Student’s t-tests; * p < 0.05, ** p < 0.01.

Figure 2

Thrombus formation on immobilized collagen peptides with or without a (GPO)_n motif. Whole blood was perfused over microspots M1 (GFOGER-GPO + VWF-BP), M2 (CRP-XL + VWF-BP), M3 (GAOGER-GPO + VWF-BP), M4 (GFOGER-GPP + VWF-BP), and M5 (VWF-BP), with assumed platelet adhesion via GPIb, GPVI, and/or integrin α₂β₁, as in Table 1. The wall-shear rate was 1000 s⁻¹, with a perfusion time of 3.5 min. Representative bright-field microscopic images at the end stage are shown for an analysis of platelet deposition (parameter P1) and thrombus characteristics (P2–5). In addition, end-stage three-color fluorescence images for an analysis of PS exposure (AF568 annexin A5, P6), CD62P expression (AF647 α-CD62P, P7), and fibrinogen binding (FITC, P8) are shown. Scale bars represent 50 μm.

Figure 3

Effect of Syk inhibition on parameters of thrombus formation on immobilized collagen peptides. Blood samples pre-incubated with vehicle (Ctrl) or Syk-IN (20 µM) flowed over microspots M1–5, and the thrombi formed were imaged to obtain parameters P1–8, as in Figure 2. The effects of Syk-IN were assessed per blood sample, surface, and parameter. Mean values from individual blood samples (n = 5–7) were univariate-scaled to 0–10 per parameter across all surfaces M1–9. (A) Heatmap of scaled parameters, demonstrating the mean effects of Syk-IN. The rainbow color code indicates scaled values between 0 (blue) and 10 (red). (B) Subtraction heatmap representing the scaled effects of Syk-IN, filtered for relevant changes (p < 0.05, paired Student’s t-test per surface and parameter). The color code represents decreases (green) or increases (red) in comparison to control runs. (C) Cumulative inhibitory effect per parameter over all microspots, indicating relevant changes.

Figure 4

Syk inhibition differently affecting platelet Ca²⁺ rises by collagens. Fura-2-loaded platelets in 96-well plates were pre-incubated with Syk-IN (5 µM) or were left untreated before stimulation with different collagens (M6–9, 10 µg/mL). Changes in [Ca²⁺]_i were continuously monitored per well-plate row by ratio fluorometry using a FlexStation 3. Collagens were injected at 60 s (arrow), and they reached platelets in a diffusion-limited way. (A) Calibrated [Ca²⁺]_i traces recorded for 600 s in the absence (black, control) or presence (gray) of a Syk inhibitor. Traces are representative of three experiments. (B) Quantification of [Ca²⁺]_i rises after 300 s (top graph) and 600 s (bottom graph) for M1–5. Means ± SEM (n = 3). Paired Student’s t-tests; * p < 0.05, ** p < 0.01.

Figure 5

Thrombus formation on immobilized collagens. Whole blood was perfused over microspots M6 (collagen-H), M7 (fibrillar collagen-I), M8 (degraded collagen-I), and M9 (collagen-III). The wall-shear rate was 1000 s⁻¹, and the perfusion time was 3.5 min. Representative bright-field microscopic images at the end stage are shown for an analysis of platelet deposition (parameter P1) and thrombus characteristics (P2–5). In addition, end-stage three-color fluorescence images for an analysis of PS exposure (AF568 annexin A5, P6), CD62P expression (AF647 α-CD62P, P7), and fibrinogen binding (FITC, P8) are shown. Scale bars represent 50 μm.

Figure 6

Effect of Syk inhibition on parameters of thrombus formation on immobilized collagen. Whole blood pre-incubated with vehicle (Ctrl) or Syk-IN (20 µM) was perfused over microspots M6–9, and thrombus formation was imaged to obtain parameters P1–8, as in Figure 5. The effects of Syk-IN were calculated per blood sample, surface, and parameter. Mean values for all blood samples (n = 5–7) were univariate-scaled to 0–10 per parameter across all surfaces of M1–9. (A) A heatmap of the scaled parameters showing the mean effects of Syk-IN. The rainbow color code gives scaled values between 0 (blue) and 10 (red). (B) A subtraction heatmap representing the scaled effects of Syk-IN, filtered for relevant changes (p < 0.05, paired Student’s t-tests per surface and parameter). The color code represents decreases (green) or increases (red) in comparison to control runs. (C) Cumulative inhibitory effect over all microspots per parameter, indicating relevant changes from control runs.

Figure 7

Schematic platelet adhesion and activation by collagen under flow or in suspension. (A) Under flow conditions, immobilized collagen-H interacted with VWF to capture platelets via GPIb-V-IX and activate platelets via GPVI and integrin α₂β₁. Thrombi built up through the recruitment of flowing platelets interacting with collagen/VWF-adhered platelets. Syk inhibition suppressed initial platelet activation and platelet aggregate formation. (B) Collagen-H added to a suspension of platelets transiently interacted with GPVI, resulting in Syk-dependent Ca²⁺ rises. Autocrine agonists stimulated non-adhered platelets, responding through Syk-independent signals.