Brazilin isolated from Caesalpinia sappan L. acts as a novel collagen receptor agonist in human platelets

Background Brazilin, isolated from the heartwood of Caesalpinia sappan L., has been shown to possess multiple pharmacological properties. Methods In this study, platelet aggregation, flow cytometry, immunoblotting analysis, and electron spin resonance (ESR) spectrometry were used to investigate the effects of brazilin on platelet activation ex vivo. Moreover, fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used in in vivo study. Results We demonstrated that relatively low concentrations of brazilin (1 to 10 μM) potentiated platelet aggregation induced by collagen (0.1 μg/ml) in washed human platelets. Higher concentrations of brazilin (20 to 50 μM) directly triggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an antagonist of ADP). It was not inhibited by yohimbine (an antagonist of epinephrine), by SCH79797 (an antagonist of thrombin protease-activated receptor [PAR] 1), or by tcY-NH2 (an antagonist of PAR 4). Brazilin did not significantly affect FITC-triflavin binding to the integrin αIIbβ3 in platelet suspensions. Pretreatment of the platelets with caffeic acid phenethyl ester (an antagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies raised against collagen receptor glycoprotein VI and integrin α2β1, respectively, abolished platelet aggregation stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the phosphorylation of phospholipase C (PLC)γ2 and Lyn, which were significantly attenuated in the presence of JAQ1 and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. An in vivo mouse study showed that brazilin treatment (2 and 4 mg/kg) significantly shortened the occlusion time for platelet plug formation in mesenteric venules. Conclusion To the best of our knowledge, this study provides the first evidence that brazilin acts a novel collagen receptor agonist. Brazilin is a plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.

Background Brazilin (7,11b-dihydrobenz[b]indeno [1,2-d]pyran-3,6a,9,10 (6H)-tetrol) is the major component isolated from the heartwood of Caesalpinia sappan L. (Leguminosae) (Figure 1). C. sappan has long been widely used as an oriental traditional or folk medicine. It is considered an analgesic and antiinflammatory agent and has been used to treat emmeniopathy, sprains, and convulsions [1]; it has also been used to treat diabetic complications [2] and to improve blood circulation [3]. Extracts of C. sappan have been shown to exert various pharmacological effects, including anti-hypercholesterolemia, sedation, and depression of the central nervous system [4]. In addition, it is an anti-hepatitis B surface antigen (HBsAg) [5] and lowers the motility of human sperm [6]. Brazilin is also used as a natural red pigment for histological staining [7]. Several studies have shown that the anti-hyperglycemic [8], anti-hepatotoxic [9], and antiinflammatory effects of brazilin are caused by the inhibition of inducible nitric oxide synthase (NOS) in macrophage cells [10], and vasorelaxation induced by the activation of NOS in endothelial cells [4].
Intravascular thrombosis is associated with several cardiovascular diseases. The initiation of an intraluminal thrombosis is thought to involve platelet adherence and aggregation. During normal circulation, platelets do not aggregate. However, when a blood vessel is damaged, platelets adhere to the disrupted surface and release biologically active constituents that induce aggregation [11]. Resting (circulating) platelets are anuclear cells, discoid in shape, which originate from megakaryocytes in the bone marrow. Platelets may be activated by various physiological or pharmacological agents. Physiological agents include thrombin, collagen, ADP, platelet-activating factor (PAF), and epinephrine, whereas pharmacological agents include calcium ionophores and cyclic endoperoxide analogues. Upon activation, the platelets lose their discoid shape and become more spherical, extending long, spiky pseudopods and bulky surface protrusions [12]. The various agonists are thought to exert their effects by interacting with specific receptors on platelet membranes. Platelet activation plays a crucial role in numerous cardiovascular and cerebrovascular disorders.
Until this study, no data had been published on the effect of brazilin in platelet activation. One study reported that brazilin significantly inhibited thrombin-, collagen-, and ADP-induced platelet aggregation in washed rat platelets [13]. By contrast, our preliminary study showed that brazilin potentiated or stimulated platelet aggregation in washed human platelets. This discrepancy might result from specie-specific characteristics of platelets. We thus systematically examined the influence of brazilin in human platelets ex vivo and in platelet plug formation in vivo. The findings were used to characterize the mechanisms of brazilin-mediated activation in human platelets.

Preparation of human platelet suspensions
Human platelet suspensions were prepared as previously described [14]. This study conformed to the principles outlined in the Helsinki Declaration, and human volunteers gave informed consent. In brief, blood was collected from healthy human volunteers who had taken no medicine during the preceding 2 wk, and was mixed with acid-citrate-dextrose (ACD) (9:1, v/v). After centrifugation, the supernatant (platelet-rich plasma; PRP) was supplemented with PGE 1 (0.5 μM) and heparin (6.4 IU/ ml) and incubated for 10 min. The mixture was then centrifuged at 500 g; thereafter, the platelets were washed and suspended in a Tyrode's solution containing BSA (3.5 mg/ml). The final concentration of Ca +2 in the Tyrode's solution was 1 mM.

Platelet aggregation
A turbidimetric method was applied to measure platelet aggregation [14], using a Lumi-Aggregometer (Payton, Canada). Platelet suspensions (3.6 × 10 8 cells/ml) were pretreated with or without reagents for 3 min, followed by the addition of brazilin or various agonists to trigger platelet activation. The reaction was allowed to proceed for at least 6 min, and the extent of aggregation was expressed in light-transmission units.

Flow cytometric analysis
Fluorescence-conjugated triflavin, an α IIb β 3 disintegrin, was prepared as previously described [11]. Platelet suspensions (3.6 × 10 8 cells/ml) were preincubated with brazilin (25 and 50 μM) or a solvent control (0.5% DMSO) for 3 min, followed by the addition of 2 μl of a solution of FITC-triflavin (2 μg/ml). The suspensions were then assayed for fluorescein-labeled platelets, using a flow cytometer (Beckman Coulter, Miami, FL). Data were collected from 50,000 platelets per experimental group, and the platelets were identified by their characteristic forward and orthogonal light-scattering profiles. All experiments were repeated at least 4 times to ensure reliability.

Immunoblotting
Washed platelets (1 × 10 9 cells/ml) were preincubated with reagents for 3 min, followed by the addition of agonists to trigger platelet activation. The reaction was stopped, and platelets were immediately re-suspended in 200 μl of lysis buffer. Samples containing 80 μg of protein were separated using a 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); proteins were electrotransferred by semidry transfer (Bio-Rad, Hercules, CA). Blots were blocked with TBST (10 mM Tris-base, 100 mM NaCl, and 0.01% Tween 20) containing 5% BSA for 1 h and then probed with various primary antibodies. Membranes were incubated with HRP-linked anti-mouse IgG or anti-rabbit IgG (diluted 1:3000 in TBST) for 1 h. Immunoreactive bands were detected using an ECL system. Bar graphs depicting quantitative ratios were produced by scanning the reactive bands and quantifying their optical density using videodensitometry (Bio-profil; Biolight Windows Application V2000.01; Vilber Lourmat, France).

Measurement of hydroxyl radicals by electron spin resonance (ESR) spectrometry
The ESR method used a Bruker EMX ESR spectrometer as described previously [15]. In brief, platelet suspensions (3.6 × 10 8 cells/ml) were incubated with brazilin (25 and 50 μM), collagen (1 μg/ml) or a solvent control (0.5% DMSO) for 3 min. The reaction was allowed to proceed for 5 min, followed by the addition of DMPO (100 μM) for the ESR study.
Fluorescein sodium-induced platelet thrombi in mesenteric microvessels of mice As described previously [14], mice were anesthetized, and an external jugular vein was cannulated with PE-10 so that dye and medication could be administered by an intravenous (i.v.) bolus. A segment of the small intestine was placed onto a transparent culture dish for microscopic observation. Venules (30 to 40 μm) were selected for irradiation to produce a microthrombus. Using the epi-illumination system, light from a 100-W mercury lamp was passed through a B-2A filter (Nikon, Tokyo, Japan) with a DM 510 dichromic mirror (Nikon). Wavelengths below 520 nm had been eliminated from the filtered light, which was used to irradiate a microvessel; the area of irradiation was approximately 100 μm in diameter on the focal plane. Various dosages of brazilin (2 and 4 mg/kg) or an isovolumetric solvent control (0.5% DMSO) was administered 1 min after fluorescein sodium (15 μg/kg) administration. Five minutes after administration of the fluorescein sodium, irradiation by filtered light and the video timer were simultaneously begun, and platelet aggregation was observed on a television monitor. The time lapse for inducing thrombus formation leading to the cessation of blood flow was measured.

Statistical analysis
The experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations. Paired Student's t-test was used to determine significant differences in the in vivo studies of platelet plug formation. The other experiments were assessed by the method of analysis of variance (ANOVA). If this analysis indicated significant differences among the group means, then each group was compared using the Newman-Keuls method. A p value of less than 0.05 was considered statistically significant.
Triflavin is an α IIb β 3 disintegrin, which inhibits platelet aggregation by directly interfering with fibrinogen binding to the integrin α IIb β 3 [11]. We evaluated whether brazilin would bind directly to the platelet integrin α IIb β 3 , leading to interruption of platelet aggregation. Our results showed that the relative intensity of the fluorescence of 2 μg/ml FITC-triflavin bound directly to platelets was 55.2 ± 4.5 ( Figure 4G, a). The fluorescent intensity was markedly reduced in the presence of 5 mM EDTA (negative control, 5.2 ± 0.6) ( Figure 4G, b). Brazilin (25 and 50 μM) did not significantly affect FITC-triflavin binding to the integrin α IIb β 3 in platelet suspensions (25 μM, 55.1 ± 5.2; 50 μM, 54.3 ± 4.5) ( Figure 4G, c and d). These results showed that the stimulatory effect of brazilin on platelet aggregation did not affect integrin α IIb β 3 . Overall, our findings provide evidences that brazilin acts as a collagen receptor agonist.

Influence of brazilin in hydroxyl radical formation in vitro and platelet plug formation in microvessels of mice
A typical ESR signal of hydroxyl radical (OH • ) formation was triggered in collagen-activated platelets compared to resting platelets or 0.5% DMSO-treated platelets ( Figures 6A,  a, b, and c). However, treatment with brazilin (25 and 50 μM) did not significantly trigger hydroxyl radical formation ( Figures 6A, d and e).
Our observation of thrombus formation in the microvessels of mice pretreated with fluorescein sodium (15 μg/kg) showed that the required occlusion time was approximately 90 s. When brazilin (2 and 4 mg/kg) was administered after pretreatment with fluorescein sodium, the occlusion time was significantly shorter compared with the solvent controls (occlusion time for 2 mg/kg brazilin was 74.4 ± 2.4 s compared with 91.9 ± 2.3 s for 0.5% DMSO; n=5, p < 0.05; for 4 mg/kg brazilin, occlusion time was 72.7 ± 3.4 s compared with 91.2 ± 3.8 s for 0.5% DMSO; n=5, p < 0.05) ( Figure 6B). These results indicated that brazilin stimulated platelet plug formation in vivo.

Discussion
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 A 2 (TxA 2 ) from the arachidonic acid (AA) or by the secretion of ADP from the dense granules in platelets. ADP binds to 2 major purinergic receptors (P2Y 1 and P2Y 12 ), which play an important role in potentiating platelet activation induced by other aggregating agonists [21]. Therefore, ATP, an antagonist to ADP, might affect platelet aggregation stimulated by other agonists, including brazilin ( Figure 2C).

Number of cells
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 [12]. Of the 4 known PAR isoforms, PAR1, PAR3, and PAR4 constitute the active thrombin receptors on human platelets [22]. PAR1 and PAR4 are essential for thrombin-induced human platelet activation [23]. Furthermore, epinephrine could induce platelet aggregation in the presence of sub-physiological calcium concentrations, as occurs in citrated plasma [24]. Aggregation as monitored in the light transmission aggregometer occurs without preceding shape change (disc to sphere transformation) ( Figure 2D). Platelets possess stimulatory α 2 -adrenoceptors and inhibitory β-adrenoceptors; in most individuals the α 2 -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 TxA 2 , whereas aggregation is entirely dependent on the release thereof [21]. 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 α 2 β 1 (GP Ia/IIa) and GP VI [19] 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 [25]. Integrin α 2 β 1 is another major collagen receptor on endothelial cells and platelets. In cells expressing integrin α 2 β 1 , many signals (including tyrosine phosphorylation and matrix remodeling) are activated after cell adhesion to collagen [26]. Recent findings suggest that integrin α 2 β 1 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 [29]. Fyn and Lyn were shown to bind to the Pro-rich domain of the GP VI cytoplasmic tail in platelets [30], 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., PLCγ 2 ). Our previous study [17] 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 PLCγ 2 and Lyn phosphorylation. All these reactions were significantly diminished by JAQ1 (anti-GP VI mAb) and Sam.G4 (anti-integrin α 2 β 1 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 [31]. A study also reported that some allosteric ligands can enhances both affinity and efficacy, and it displays allosteric agonism [31]. 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 Ca 2+ during the initial phase of platelet activation processes [15]. 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 [15]. 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 [11]. When platelets aggregate, they release a number of substances including TxA 2 and ADP, both of which strengthen the platelet activation processes. He et al. [27] showed that integrin α 2 β 1 -deficient mice exhibited delayed thrombus formation following carotid artery injury. This result was consistent with the previously reported correlation between high levels of integrin α 2 β 1 expression and increased risk for thrombosis involving the coronary and cerebral vessels [32,33]. Nieswandt et al. [34] 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.

Conclusions
In conclusion, the key finding of this study was that brazilin acts as a collagen receptor agonist. However, the detailed mechanisms of brazilin-mediated signaling events in platelet activation require further investigation. Brazilin is a novel plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.