In this study, up to our knowledge, this is a novel finding that brazilin, a plant-based natural product acts as a collagen receptor agonist induce platelet activation, other than that some collagen receptor agonists purified from the snake venoms [19, 20].
Platelets are activated by a variety of physiological stimuli (e.g., thrombin, collagen, ADP, epinephrine, and PAF). These agonists are thought to exert their effects by interacting with specific receptors on the platelet membranes. The primary effects of agonists may be enhanced by secondary effects caused by the synthesis of thromboxane A2 (TxA2) from the arachidonic acid (AA) or by the secretion of ADP from the dense granules in platelets. ADP binds to 2 major purinergic receptors (P2Y1 and P2Y12), which play an important role in potentiating platelet activation induced by other aggregating agonists . Therefore, ATP, an antagonist to ADP, might affect platelet aggregation stimulated by other agonists, including brazilin (Figure 2C).
Thrombin is one of the most potent activators of platelets and its role in promoting thrombus formation has been clearly established. Thrombin activates platelets through multiple cell-surface receptors, including the GP Ib/V/IX complex and the PARs . Of the 4 known PAR isoforms, PAR1, PAR3, and PAR4 constitute the active thrombin receptors on human platelets . PAR1 and PAR4 are essential for thrombin-induced human platelet activation . Furthermore, epinephrine could induce platelet aggregation in the presence of sub-physiological calcium concentrations, as occurs in citrated plasma . Aggregation as monitored in the light transmission aggregometer occurs without preceding shape change (disc to sphere transformation) (Figure 2D). Platelets possess stimulatory α2-adrenoceptors and inhibitory β-adrenoceptors; in most individuals the α2-adrenoceptors predominate.
Platelets adhere to the connective tissue protein collagen, with a resulting change in shape and the release of granules. Adhesion is partly dependent on the release of ADP and TxA2, whereas aggregation is entirely dependent on the release thereof . The matrix protein collagen is present in the vascular subendothelium and vessel wall, and acts as a substrate for platelet adhesion; it is also an endogenous platelet activator. Among the platelet receptors known to interact directly with collagen, integrin α2β1 (GP Ia/IIa) and GP VI  appear to play a key role and have recently gained the attention of researchers. GP VI is widely recognized as a requisite factor for the formation of platelet aggregates on a collagen surface under blood flow . Integrin α2β1 is another major collagen receptor on endothelial cells and platelets. In cells expressing integrin α2β1, many signals (including tyrosine phosphorylation and matrix remodeling) are activated after cell adhesion to collagen . Recent findings suggest that integrin α2β1 and GP VI might contribute to the overall processes of platelet adhesion and activation [19, 27, 28].
GP VI is a platelet membrane protein with a molecular weight of 62 kDa. It has been identified as a physiological collagen receptor and belongs to a membrane of the immunoglobulin superfamily, which forms a complex with the Fc receptor γ-chain (FcRγ) containing immunoreceptor tyrosine-based activation motifs (ITAM) and is phosphorylated by Src-family kinases such as Fyn and Lyn [16, 25]. Tyrosine kinases (Fyn and Lyn) are involved in GP VI-dependent activation and might phosphorylate the FcRγ . Fyn and Lyn were shown to bind to the Pro-rich domain of the GP VI cytoplasmic tail in platelets , suggesting that the GP VI-dependent activation mechanism might be similar to that of the cytokine receptors. In this process, receptor-bound tyrosine kinases (such as Src) phosphorylate the cytoplasmic tails of receptors when the receptors become associated with each other through ligand binding. This phosphorylation will initiate the signal transduction pathway. In platelets, cross-linking of the GP VI/FcRγ complex would enable the GP VI-bound Fyn or Lyn to move to a position close enough to FcRγ that it would catalyze the phosphorylation of FcRγ ITAM. In turn, this triggers the phosphorylation of downstream signals, including the linker for activation of T-cells (LAT), leading to the activation of a kinase cascade (i.e., PLCγ2).
Our previous study  showed that the antiplatelet activity of CAPE might involve direct interference with the binding of collagen to its specific receptors on the platelet membrane. The current study showed that CAPE markedly inhibited brazilin-induced platelet aggregation. Furthermore, brazilin markedly stimulated platelet aggregation and PLCγ2 and Lyn phosphorylation. All these reactions were significantly diminished by JAQ1 (anti-GP VI mAb) and Sam.G4 (anti-integrin α2β1 mAb). Interestingly, we also found that the relative fluorescence intensity of the FITC-collagen (1 μg/ml) bound directly to platelets was 11.7 ± 1.9 (n=4) and hence the fluorescent intensity was markedly reduced in the presence of 1 μg/ml collagen (1.6 ± 1.4, n=4); however pretreatment with brazilin (25, 50, and 100 μM) showed a significant increase in the relative fluorescence intensity of FITC-collagen (25 μM, 33.8 ± 13.9; 50 μM, 38.4 ± 10.6; 100 μM, 61.8 ± 9.8; n=4) (data not shown). These results suggest that brazilin may act at the allosteric site to display allosteric agonism on collagen receptors, and subsequently enhances both the affinity and efficacy of collagen towards its binding sites. A similar model has been proposed in G-protein-coupled receptors and predicts that allosteric ligands bind to a topographically distinct site on a receptor to modulate orthosteric ligand affinity and/or efficacy . A study also reported that some allosteric ligands can enhances both affinity and efficacy, and it displays allosteric agonism . Therefore, we speculate that brazilin may serve as an allosteric ligand for collagen receptors in platelets. Overall, these results provided evidence that the stimulation of platelet activation by brazilin might be the result of direct stimulation of collagen receptors on the platelet membrane. However, our experiments did not rule out the possibility that other as-yet-unidentified mechanisms might be involved in brazilin-mediated platelet activation.
Reactive oxygen species (i.e., hydrogen peroxide and hydroxyl radicals) derived from platelet activation might amplify platelet reactivity during in vivo thrombus formation. Free radical species act as secondary messengers that increase cytosolic Ca2+ during the initial phase of platelet activation processes . It is also evident that some of the hydrogen peroxide produced by platelets is converted into hydroxyl radicals, as platelet aggregation can be inhibited by hydroxyl radical scavengers . In the present study, we found that brazilin did not significantly induce hydroxyl radical formation as compared with the collagen-stimulated platelets, indicating that brazilin may have a differential characterization on free radical formation apart from acting as the collagen receptor agonist in platelets. Following an injury to the endothelial cells, exposure of sub-endothelial collagen provides the major trigger to initiate platelet adhesion and aggregation at the site of injury. This is followed by arterial thrombus formation . When platelets aggregate, they release a number of substances including TxA2 and ADP, both of which strengthen the platelet activation processes. He et al.  showed that integrin α2β1-deficient mice exhibited delayed thrombus formation following carotid artery injury. This result was consistent with the previously reported correlation between high levels of integrin α2β1 expression and increased risk for thrombosis involving the coronary and cerebral vessels [32, 33]. Nieswandt et al.  reported that mice depleted of GP VI were completely protected from lethal collagen-induced pulmonary thromboemboli. Similarly, our study showed that brazilin potentiated platelet plug formation in the mesenteric venules of rats. Activated platelets also contribute to enhance the assembly and activity of two major coagulation factor complexes which facilitates coagulation and thrombus stabilization. Therefore, the coagulation factors may be involved in brazilin shortened the occlusion time in vivo.