Glycoprotein IIb/IIIa and P2Y12 induction by oligochitosan accelerates platelet aggregation

Abstract

Platelet membrane receptor glycoprotein IIb/IIIa (gpiibiiia) is a receptor detected on platelets. Adenosine diphosphate (ADP) activates gpiibiiia and P2Y12, causing platelet aggregation and thrombus stabilization during blood loss. Chitosan biomaterials were found to promote surface induced hemostasis and were capable of activating blood coagulation cascades by enhancing platelet aggregation. Our current findings show that the activation of the gpiibiiia complex and the major ADP receptor P2Y12 is required for platelet aggregation to reach hemostasis following the adherence of various concentrations of chitosan biomaterials [7% N,O-carboxymethylchitosan (NO-CMC) with 0.45 mL collagen, 8% NO-CMC, oligochitosan (O-C), and oligochitosan 53 (O-C 53)]. We studied gpiibiiia and P2Y12 through flow cytometric analysis and western blotting techniques. The highest expression of gpiibiiia was observed with Lyostypt (74.3 ± 7.82%), followed by O-C (65.5 ± 7.17%). Lyostypt and O-C resulted in gpiibiiia expression increases of 29.2% and 13.9%, respectively, compared with blood alone. Western blot analysis revealed that only O-C 53 upregulated the expression of P2Y12 (1.12 ± 0.03-fold) compared with blood alone. Our findings suggest that the regulation of gpiibiiia and P2Y12 levels could be clinically useful to activate platelets to reach hemostasis. Further, we show that the novel oligochitosan is able to induce the increased expression of gpiibiiia and P2Y12, thus accelerating platelet aggregation in vitro.

Figure 1

In vitro expression levels of gpiibiiia upon the adherence of chitosan biomaterial. Depicted is the average of the mean ± SEM of fourteen fluorescence measurements, as obtained by flow cytometry.

Figure 2

Example of gpiibiiia expression levels demonstrated after the adherence of chitosan biomaterials. The expression levels of gpiibiiia are depicted in the dot plot results (CD 41a FITC-A × CD 61 PerCP-A), and the gpiibiiia-positive population is expressed in Q2 region. The events and percentages of each tested biomaterial are indicated by the red boxes. The same gating and parameter settings (FITC × PerCP) were used for all tests (n = 14). (a) 7% NO-CMC; (b) 8% NO-CMC; (c) O-C; (d) O-C 53; (e) Lyostypt; and (f) blood alone.

Figure 3

Expression of P2Y₁₂ upon the adherence of chitosan biomaterials in vitro. Protein was extracted from PRP exposed to chitosan, and the expression of P2Y₁₂ was determined by western blotting (Figure 3(a)). The protein bands were visualized using an image analyzer and band density was analyzed using ImageJ software, with the fold-change differences in expression compared with blood alone written below each band. Figure 3(b). The expression levels of P2Y₁₂ upon the presence of chitosan biomaterials are shown as the fold change in comparison with blood alone (1-fold). The blots shown are representative of the mean ± SEM from three independent experiments (n = 3). The dotted line depicts the expression level in the blood alone (1-fold).