Canine platelets express functional Toll-like receptor-4: lipopolysaccharide-triggered platelet activation is dependent on adenosine diphosphate and thromboxane A2 in dogs

Background Functional Toll-like receptor 4 (TLR4) has been characterized in human and murine platelets indicating that platelets play a role in inflammation and hemostasis during sepsis. It is unclear whether canine platelets could express functional TLR4 by responding to its ligand, lipopolysaccharide (LPS). We sought to determine if dogs express functional TLR4 and if LPS-induced platelet activation requires co-stimulation with ADP or thromboxane A2 (TxA2). Canine platelets were unstimulated (resting) or activated with thrombin or ADP prior to flow cytometric or microscopic analyses for TLR4 expression. We treated resting or ADP-primed platelets with LPS in the absence or presence of acetylsalicylic acid (ASA) and inhibited TLR4 with function blocking antibody or LPS from Rhodobacter sphaeroides (LPS-RS). Results We discovered that dog platelets have variable TLR4 expression, which was upregulated following thrombin or ADP activation. LPS augmented P-selectin expression and thromboxane B2 secretion in ADP-primed platelets via TLR4. Inhibition of cyclooxygenase by ASA attenuated LPS-mediated P-selectin expression demonstrating that TLR4 signaling in platelets is partially dependent on TxA2 pathway. Conclusion Expression of functional TLR4 on canine platelets may contribute to hypercoagulability in clinical septic dogs. Cyclooxygenase and TxA2 pathways in TLR4-mediated platelet activation may present novel therapeutic targets in dogs with sepsis.


Background
Despite recent advances in veterinary medicine, mortality remains high in dogs with sepsis secondary to Gram negative bacterial infections [1][2][3]. One of the major components of the outer membrane of Gram negative bacteria is the endotoxin, lipopolysaccharide (LPS), whose receptor, Toll-like receptor 4 (TLR4), is present on the surface of a wide variety of immune cells such as dendritic cells, epithelial cells, polymorphonuclear cells and macrophages. Formation of the TLR4 receptor complex in response to LPS initiates signalling pathways leading to proinflammatory cytokine production and inflammatory response. In addition to being the primary effector cell in hemostasis, there is growing evidence demonstrating that platelets function as innate immune cells [4]. Bacteria like E.coli and Streptococcus directly interact with platelets leading to platelet activation and aggregation [5]. Murine and human platelets also express several Toll-like receptors (TLRs), suggesting that platelets can act as sentinel cells in detecting pathogen-associated molecular patterns (PAMPs) like LPS.
Thrombocytopenia, a common finding in septic dogs, is associated with mortality, though the exact mechanism of this hematologic abnormality is poorly understood [6][7][8]. Proposed mechanisms of sepsis-associated thrombocytopenia include decreased thrombopoiesis and increased platelet consumption and sequestration. Systemic platelet activation, which precedes platelet accumulation in organs and microvasculature in human septic patients, suggests that platelets may be the key effector for systemic coagulation during bacterial infection [9]. Systemic hypercoagulability could progress to disseminated intravascular coagulation, further impeding blood flow to tissues causing organ dysfunction. Andonegui et al. showed that platelet TLR4 is an important regulator of endotoxin-mediated thrombocytopenia in mice [10]. In another in vivo sepsis model, transfusion of TLR4 deficient platelets in platelet-depleted mice attenuated microvascular thrombosis [11]. These studies suggest that platelet TLR4 also may play a role in facilitating platelet activation in sepsis leading to microvascular thrombosis, and organ dysfunction in septic dogs. [10,11] This, however, has never been demonstrated in this species. In one of the few canine studies, Yilmaz et al., demonstrated increased platelet aggregation in a lethal endotoxin shock model [8]. Another study, however, found that circulating platelets in dogs with septic peritonitis have decreased aggregation in response to multiple agonists [12].
The mechanism of platelet activation in sepsis has been extensively studied in mice and humans with conflicting results. While some investigators found that LPS stimulates human platelets to undergo activation and aggregation, others found that LPS does not directly stimulate platelets or that LPS-triggered activation requires synergistic stimulation by platelet agonists like ADP, collagen and thromboxane A 2 (TxA 2 ) [5,[13][14][15][16]. Because platelet activation mediated by TLR4 may account for the interplay between sepsis and thrombosis in dogs, a better understanding of platelet TLR4 expression and platelet response to LPS, is needed. We, therefore, aimed to examine platelet membrane TLR4 expression and determine if this expression is altered by the platelet agonists, ADP and thrombin. We also aimed to determine if LPS could activate platelets via TLR4. Specially, we sought to determine if LPS, in the absence or presence of ADP or TxA 2 , could stimulate platelet alpha-granule secretion. Lastly, we sought to determine if inhibition of platelet TLR4 could attenuate platelet response to LPS in the absence or presence of platelet priming by ADP.

Results
Out of the 30 dogs studied, 14  Canine platelets express surface TLR4 and its expression is upregulated by thrombin and ADP Resting platelets had a low surface expression of TLR4 (9.50%; IQR = 0.70-16.88) and the expression was highly variable among subjects with a coefficient of variation (CV) of 135.54%. Thrombin or 10 μM ADP significantly increased the number of TLR4-positive platelets relative to resting platelets (20.80%, IQR = 5.39-43.43, p = 0.0078; 12.12%, IQR = 1.31-45, p = 0.016, respectively) ( Fig. 1a to d). CVs of TLR4 expression in thrombin-and ADP-stimulated platelets were 85 and 97.62%, respectively. ADP increased TLR4 expression in a dosedependent manner (p = 0.047) (Fig. 1d). Compared to ADP-stimulated platelets, thrombin stimulation resulted in higher TLR4 MFI fold change (0.065 ± 0.064 vs. 0.14 ± 0.12), but this difference did not reach statistical significance (p = 0.078). Flow cytometry findings were confirmed by directly visualizing surface TLR4 expression using confocal and STED immunofluorescence microscopy. The platelet membrane was identified by detecting the highly expressed platelet integrin subunit, β3 (Fig. 2). As expected, resting platelets have minimal exteriorization of P-selectin and TLR4 was either sparcely expressed or expressed in clusters on the cell membrane of unpermeabilized platelets (Fig. 2a, arrow). Following ADP or thrombin-induced activation, surface expression of TLR4 and P-selectin was upregulated on the platelet membrane. Compared to the clustered conformation seen on resting platelets, TLR4 was distributed evenly on the membrane surface (Fig. 2b, c). In addition, co-localization of TLR4 and P-selectin on the membrane surface was detected on either ADP or thrombin-stimulated platelets (Fig. 2b, c). We then determined the location of intracellular TLR4 by permeabilizing fixed resting platelets prior to immunostaining. We found that TLR4 (Fig. 2d, Merge, arrows) was concentrated within the platelet alpha-granules as outlined by P-selectin ( Fig. 2d, P-selectin, asterisks).

Platelet priming by ADP potentiates LPS-mediated alphagranule secretion
To determine if ADP could augment platelet response to LPS, we first primed platelets with 10 μM ADP, followed by 1, 5 or 10 μg/ml LPS. Compared to platelets treated with 5 μg/ml LPS alone, ADP priming prior to treatment with LPS of the same concentration resulted in significant elevation in P-selectin (20.51% ± 17.44 vs. 33.20% ± 19.83, p = 0.0032; MFI fold change 0.044 ± 0.047 vs. 0.18 ± 0.13, p = 0.0092) (Fig. 4b, c). ADP-priming prior to LPS stimulation also led to significant increase in Pselectin expression compared to platelets activated with ADP alone (MFI fold change: 0.18 ± 0.13 vs. 0.13 ± 0.14, Fig. 1 Platelet activation upregulates surface TLR4 expression. Platelet surface TLR4 expression was measured on isolated platelets from 10 dogs using flow cytometry. a,c Representative histograms of resting (unstimulated) platelets (grey) and activated platelets (clear) indicating increase in surface TLR4 expression following thrombin (a) and ADP (c) activation. b Thrombin stimulation led to significant increase in percentage (%) of TLR4-positive platelets compared to resting platelets. d Platelets stimulated with 10 μM ADP had significantly higher percentage of TLR-positive platelets. First and third quartiles were represented by the lower and upper boundaries, respectively. + and the line within the box represents the mean and median, respectively. Whiskers represent the range of data. * p < 0.05 p = 0.038) (Fig. 4c). When ADP-primed platelets were treated with 10 μg/ml LPS, P-selectin MFI fold was significantly higher compared to unprimed platelets treated with the same concentration of LPS (MFI fold change 0.11 ± 0.070 vs. 0.0064 ± 0.08, p = 0.00020) but this elevation did not differ from that in ADP-treated platelets (p = 0.059) (Fig. 4c). ADP-priming followed by LPS stimulation significantly increased the number of P-selectin-positive platelets relative to ADP-activated platelets (35.01% ± 20.48 vs 29.88% ± 19.70, p = 0.035) and unprimed platelets treated with 1 μg/ml LPS (35.01% ± 20.48 vs 16.82% ± 12.65, p = 0.013) (Fig. 4b).
TLR4 expression (MFI fold change) in resting platelets did not correlate with LPS-induced P-selectin expression (MFI fold change) in unprimed platelets (r = 0.25, r 2 = 0.065, p = 0.51). Following ADP priming, however, TLR4 Representative confocal and super resolution immunofluorescence microscopy demonstrating TLR4 expression on a resting, b ADP-, c thrombinactivated and d permabilized resting canine platelets. a-c Following activation, platelets were fixed and stained for TLR4 (red), P-selectin (green) and the integrin β3 (blue). a In the absence of platelet agonists, resting platelets as outlined by the abundant integrin β3 show limited to no expression of Pselectin on the membrane surface. Note the aggregregated appearance of exteriorized TLR4 (arrow) b,c In ADP-and thrombin-activated platelets, TLR4 expression is upregulated and is evently distributed across the membrane surface. Note the formation of pseudopodia and colocalization of TLR4 and Pselectin (arrowheads) on activated platelets c Extensive platelet aggregation can be seen in thrombin-activated platelets. Scale bar = 5 μm. Original magnification 100x d Resting platelets were fixed, permabilized and stained for TLR4 (red) and P-selectin (green). A single z-plane is shown here to demonstrate the presence of alpha-granules within a platelet (asterisk). Intracellular TLR4 can be seen within the alpha-granules (arrows). Scale bar = 4 μm. Experiment was replicated twice from platelets isolated from 2 dogs expression was positively and moderately correlated with LPS-mediated P-selectin expression (r = 0.70, r 2 = 0.49, p = 0.036).

LPS-mediated alpha granule secretion requires TxA 2
To determine whether TxA 2 was required for LPSmediated alpha granule secretion, we first treated platelets with ASA prior to LPS stimulation in the absence or presence of ADP. We found that ASA did not significantly affect the number of P-selectin-positive platelets in resting (ASA: 8.06%, IQR:6.38-16.12 vs. Resting: 7.87%, IQR: 6.94-17.54, p = 0.84) (Fig. 5b)  In unprimed platelets treated with LPS, number of Pselectin positive platelets was significantly lower in those with ASA treatment compared to those without (10.54%,  (Fig. 5b,c).
Inhibition of TLR4 with LPS-RS, a TLR4 antagonist, had no attenuating effect on LPS-mediated P-selectin expression in unprimed platelets. However, in the presence of ADP, LPS-RS significantly lowered LPSmediated P-selectin expression. Compared to vehicle Fig. 4 ADP-priming augments LPS-mediated alpha-granule secretion in canine platelets. Platelet alpha-granule secretion was assessed by surface P-selectin (CD62P) measured as percent (%) positive or mean fluorescence intensity (MFI) fold change on isolated platelets from 10 dogs using flow cytometry. Thrombin-stimulated platelets served as positive control. a Platelets were treated with 0, 1, 5, or 10 μg/ml LPS. Only platelets treated with 5 μg/mL LPS had significant increase in CD62P+ platelets compared to unstimulated platelets. b,c Platelets were primed with 10 μM ADP prior to stimulation with 0, 1, 5 or 10 μg/ ml LPS. LPS stimulation with 1 μg/mL LPS in ADP-primed platelets significantly elevated the percentage of CD62P+ platelets b but did not increase MFI fold change c compared to ADP-primed platelets without LPS. LPS at 5 μg/mL significantly increase CD62P MFI fold change in ADP-primed platelets relative to those without LPS. ADP priming increased platelet response to LPS at 1 (b), 5 μg/mL (b,c) and 10 μg/mL (c). First and third quartiles were represented by the lower and upper boundaries, respectively. + and the line within the box represents the mean and median, respectively. Whiskers represent the range of data. * p < 0.05, # All treatments were significantly different (p < 0.05) from positive control control, TLR4 inhibition by LPS-RS significantly decreased the percentage of P-selectin positive platelets (19.71% ± 10.86 vs 10.19% ± 4.51, p = 0.0003) and MFI fold change (0.20 ± 0.17 vs 0.099 ± 0.13, p = 0.0049). (Fig. 6c).

Discussion
To the authors' knowledge, this is the first study documenting the expression of functional TLR4 in canine platelets. The present study indicates that platelet response to E.coli LPS via TLR4 is amplified by the agonists ADP and TxA 2 in dogs.
The expression of functional TLR4 on canine platelets highlights a highly conserved mechanism of pathogen recognition utilized by many other cell types in mammals [17]. But unlike other immune cells, thrombin and ADP, which are platelet agonists that stimulate platelets in extending platelet plug formation, upregulate the surface expression of platelet TLR4 [18]. Here, we found that TLR4 resides within the cytoplasm and alpha granules in unstimulated platelets, similar to the findings in human platelets. The upregulation of surface TLR4 in thrombin-activated human platelets is secondary to the activation of calpain with subsequent cleavage of myosin-9 resulting in TLR4 trafficking from the alphagranules to the platelet plasma membrane [19]. The coexpression of TLR4 and P-selectin on the surface of activated canine platelets suggests that TLR4 trafficking to the platelet surface also could be mediated by alphagranule secretion. This unique mechanism of TLR4 upregulation in platelets highlights the interplay between hemostasis and innate immunity.
Our results indicate a highly variable expression of surface TLR4 among dogs which is augmented once Fig. 5 LPS amplifies ADP-mediated thromboxane B 2 (TxB 2 ) secretion and inhibition of platelet cyclooxygenase 1 attenuates LPS-mediated alpha-granule secretion. a Platelet TxB 2 concentration was measured by ELSIA from platelet supernatant in 10 dogs. In the presence or absence of ADP, platelets were treated with 5 μg/ml LPS. Thrombinstimulated and acetyl salicylic acid (ASA)-treated platelets served as positive and negative controls, respectively. LPS-treated platelets did not augment TxB 2 production compared to unstimulated platelets and ADP-treated platelets. ADP priming in LPS-treated platelets led to more TxB 2 than ADP-treated platelets, LPS-treated platelets and unstimulated platelets. b,c Isolated platelets from 10 dogs were pretreated with 100 μM acetylsalicyclic acid (ASA) prior to treatment with 5 μg/ml LPS with or without ADP. Platelet alpha-granule secretion was assessed by P-selectin (CD62P) measured as percent (%) positive b or mean fluorescence intensity (MFI) fold change c using flow cytometry. ASA significantly decreased the % P-selectin positive platelets a but not P-selectin MFI fold change b in LPStreated platelets in the absence or presence of ADP-priming. First and third quartiles were represented by the lower and upper boundaries, respectively, and the line within the box represents the median. + represents the mean. Whiskers represent the range of date. * p < 0.05, # Significance between all treatments and controls (p<0.05) platelets were activated by thrombin or ADP. The significant correlation between TLR4 expression and the degree of LPS-mediated alpha-granule release in the presence of ADP suggests that the upregulation of TLR4 augments platelets' sensitivity to LPS. We further confirmed this finding by inhibiting TLR4 in ADP-primed platelets. While TLR4 inhibition did not interfere with ADP-induced activation, it abolished the stimulatory effects of LPS indicating that ADP potentiates LPSmediated platelet activation by upregulating surface TLR4 expression. Dogs with naturally occurring sepsis are found to be in a hypercoagulable state with elevated thrombin generation and overconsumption of endogenous anticoagulants like antithrombin III [20]. It is unknown at this stage if elevated levels of thrombin in sepsis could trigger upregulation of surface TLR4 expression in canine platelets and, thereby, increases platelet response to endotoxins. In people, platelet TLR4 expression is elevated during sepsis and this upregulation is associated with the severity of sepsis-induced thrombocytopenia [21]. The prognostic and diagnostic significance of platelet TLR4 expression in dogs requires further investigations.
We demonstrated that E.coli LPS, to a limited extent, activates canine platelets to undergo alpha-granule secretion required for normal thrombus formation. Pselectin, a marker of alpha-granule secretion, is an integral protein of the alpha granule membrane. In accordance with previous studies, we were unable to detect significant elevation in P-selectin expression, and TxA 2 generation elicited by LPS [16]. Despite finding a significant increase in the numbers of platelets expressing Pselectin, the lack of MFI fold change suggests that LPS had minimal effect on augmenting P-selectin density, a marker of substantial alpha-granule secretion. Another explanation is the variable P-selectin expression on unstimulated platelets potentially due to stress from handling, excitement or in vitro platelet activation from PRP generation (Fig. 3) [22]. This likely could decrease platelet sensitivity to LPS. Dogs that were easily excitable, Fig. 6 LPS-mediated alpha-granule secretion is dependent on platelet TLR4. Platelet P-selectin (CD62P) measured as percent (%) positive platelets or mean fluorescence intensity (MFI) fold change on isolated platelets (1 × 10 7 /ml) from 10 dogs using flow cytometry. a, b Platelets were treated with either 50 μg/ml TLR4 function blocking antibody or 50 μg/ml IgG2a before LPS stimulation (5 μg/ml) in absence or presence of ADP. TLR4 inhibition in LPS-treated had minimal effect on attenuating the numbers of P-selectin positive platelets (a) but significantly decreased P-selectin MFI fold change b in the presence or absence of ADP priming. Pretreatment of platelets with LPS-RS before LPS stimulation significantly decreased number of P-selectin postive platelets and MFI fold change only in ADP-primed platelets. First and third quartiles were represented by the lower and upper boundaries, respectively, and the line within the box represents the median. Whiskers represent the range of data. + represents the mean * p < 0.05, #Significance between all treatments and isotype controls (p<0.05) stressed and difficult to restrain for blood draws were later on excluded from the TLR4 blocking experiments, as evidenced by the lower mean resting P-selectin expression (Fig. 5a, c).
Once platelets are primed with ADP, treatment of platelets with LPS potentiates alpha-granule secretion and TxA 2 generation, suggesting that LPS synergizes with ADP in amplifying platelet response to LPS. This observation might be due to several underlying mechanisms. First, surface P-selectin on human platelets has been shown to enhance E.coli LPS binding to platelet TLR4 by forming TLR4/CD62P receptor complex [23]. Secondly, besides upregulating surface TLR4 on platelets, ADP, which binds to the G-protein coupled receptors, P2Y1 and P2Y12, triggers signaling events critical for robust platelet activation to occur. For example, in murine and human platelets, P2Y1, which couples with G q , activates phospholipase C (PLC) and, subsequently, 1,4,5-triphosphate (IP 3 ) and diacylglycerol, to increase calcium mobilization and cytosolic calcium concentration, an event required to trigger numerous cellular events including integrin activation. Calcium mobilization via PLCγ2 also is required for TLR4 signalling in immune cells like macrophages and dendritic cells [24]. Whether calcium flux orchestrated by ADPmediated signaling amplifies TLR4 downstream signaling in canine platelets requires further investigation. In addition to alpha-granule secretion, we also found that LPS and ADP synergistically increase production of TxA 2 , an eicosanoid converted from arachidonic acid within the cell membrane by the enzyme, cyclooxygenase-1. This suggests that TLR4 and ADP receptor signaling share a similar downstream pathway that results in TxA 2 generation. Nocella et al. proposed that activation of TLR4 and ADP receptors results in amplification of the AKT- Fig. 7 Schematic diagram of LPS-mediated platelet activation and TLR4 expression in canine platelets. ADP activation via P2Y1 or P2Y12 receptor upregulates surface TLR4 expression. TLR4 trafficking to cell membrane from granules may be mediated by alpha-granule secretion. LPS binding protein (LBP) presents LPS to CD14 forming a heterodimeric complex with TLR4 and myeloid differentiation protein 2 (MD-2). Downstream signaling pathway of TLR4 leads to α-granule secretion, which is amplified by ADP and thromboxane A 2 (TxA 2 ). Activation of G-protein coupled receptors, P2Y1/ P2Y12 and thromboxane receptor (TP), leads to phospholipase C (PLC) activation and, subsequently, 1,4,5-triphosphate (IP 3 ) and diacylglycerol (DAG) for intracellular calcium release and alpha-granule secretion. LPS acts synergistically with ADP to increase generation of TxA 2 , serving a positive feedback mediator. TLR4 and ADP signaling activates cyclooxygenase-1 (COX-1), which converts arachidonic acid (AA) to TxA 2 , likely by the Akt/p38 MAPK pathway p38MAP kinase axis leading to phospholipase A2 phosphorylation and subsequent generation of TxA 2 in human platelets [16]. Further studies are needed to characterize the non-genomic pathway of TLR4 in dog platelets (Fig. 7).
By directly inhibiting the enzyme, cyclo-oxygenase (COX-1) with ASA, we showed that platelet activation mediated by TLR4 and ADP signaling is partially dependent on TxA 2 . Given its short half-life (~30 s), TxA 2 acts as an autocrine or paracrine to nearby platelets ampliflying platelet activation via platelet TxA 2 receptors. As expected, ASA did not completely attenuate LPSmediated alpha granule secretion and, in some dogs, had no effect on P-selectin expression. This may be due to the variable thromboxane responsiveness found in some dogs, in which inhibition of TxA 2 production may not negatively impact LPS-mediated platelet activation [25]. This also may be due to the presence of other mediators likely involved in the positive feedback mechanism of TLR4 signaling. Antiplatelet therapy is the cornerstone of antithrombotic therapy in dogs. In addition to their antithrombotic properties, antiplatelet drugs have been shown to modulate inflammation in people and dogs by reducing acute phase response and proinflammatory biomarkers like C-reactive protein [26]. Since TxA 2 serves as a positive feedback in amplifying LPS-mediated platelet activation, antiplatelet therapy may have potential benefits in clinical septic dogs [26]. In observational studies, prehospital administration of ASA has been shown to be associated with reduced mortality and lower prevalence of acute respiratory distress syndrome in critically ill humans with sepsis [27,28]. Prospective clinical trials are needed to identify the clinical benefits of ASA therapy in at-risk dogs.
The present study has several limitations. First, the concentration of LPS that consistently activated canine platelets in the absence or presence of ADP was 5 μg/ml, a higher concentration than expected in systemic circulation during sepsis. A plausible explanation for the need of this concentration is the low levels of plasma proteins in our washed platelet system. The binding of LPS to TLR4 is complex and requires 3 other extracellular proteins including LPS binding protein (LBP), CD14, and myeloid differentiation protein 2 (MD-2). LBP, a soluble acutephase protein, binds to LPS and presents it to CD14 on platelets to form a heterodimeric complex with TLR4 and its accessory protein, MD2 (Fig. 6). Whether human platelets express CD14 is controversial [13,29,30]. Damien et al. found that the response to LPS in washed platelets is dependent on soluble CD14 suggesting that platelets may obtain CD14 from systemic circulation [30]. We supplemented canine platelets with a small concentration of canine serum in order to enhance platelet response to LPS. However, we did not investigate if canine platelets express the necessary TLR4 signaling complex including surface CD14 expression. Since LPS-RS antagonizes LPS by directly competing with LPS for the binding site on MD-2, our data suggests that dog platelets may express MD-2 on the cell surface. Further studies using platelet rich plasma to determine if concentrations of LPS found in septic dogs could activate platelets is needed. Another plausible explanation is that canine platelets may have variable responses to LPS from different strains of E. coli as the affinity for TLR4 on mouse platelets is dependent on the LPS serotypes [23]. Finally, we did not investigate the effects of LPS on other markers of platelet activation such as fibrinogen binding, dense granule secretion and CD40L expression, which have all been shown to increase by LPS stimulation in human and murine platelets [13,23].

Conclusion
Our study demonstrated that canine platelets express functional TLR4, which can be upregulated by thrombin or ADP. Although E coli LPS is a limited stimulus for platelet activation, in the presence of ADP or TxA 2 , LPS is a potential platelet activator in dogs. The findings of this study provide novel insights into the mechanisms of thrombosis and potential therapeutic targets in septic dogs.

Animals
The study protocol was approved by the Institutional Animal Care and Use Committee at the University of California, Davis. Thirty clinically healthy staff-or student-owned dogs greater than 5 kg were used in this study and 8 dogs were enrolled for each experiment. Dogs were deemed to be clinically healthy by physical examination performed by the corresponding author and a complete blood count using an automated hematology analyzer (Coulter ACT diff, Beckman-Coulter Inc., Miami, FL) and blood smear evaluation. Dogs were not enrolled in the study if they were vaccinated 30 days prior to enrollment, on any concurrent medications, or had any abnormalities on hematological examination. Eight dogs were enrolled.

Generation of gel-filtered platelets
Whole blood (4 to 6 ml) was drawn from either the jugular or cephalic vein using a 22 gauge needle connected to a 6 ml syringe before transferring into 3.2% sodium citrate tubes. Blood tubes were gently inverted 2 to 3 times and carefully inspected for clots. Citrated blood was transferred to polypropylene tubes and platelet rich plasma was generated by centrifugation (300 x g, 5 min, no brakes) at room temperature. Platelets were separated from plasma by gel-filtration over a Sephrose 2B column at 37°C and eluted with filtered Tyrodes-HEPES buffer (pH 7.4, 5 mM dextrose, 0.5% canine serum, without divalent cations) [31]. Gelfiltered platelets were observed to determine if they exhibited the "swirling" characteristic found in truly discoid resting platelets [31,32]. Isolated platelets that failed to display swirling movements were not included in the study. Platelet count was obtained using an automated analyzer (Coulter ACT diff, Beckman-Coulter Inc., Miami, FL) and confirmed by bloodsmear evaluation. All experiments were carried out in a sterile manner.
Fluorescent images were acquired using a combination of confocal and super-resolution stimulated emission depletion (STED) microscopy (Leica TCS SP8 STED 3x, Leica Microsystems, Buffalo Grove, IL). Imaging powers of STED wavelengths were set to 20 to 50% of excitation wavelengths. The following imaging sequence was