Sulfatides Activate Platelets Through P-Selectin and Enhance Platelet and Platelet–Leukocyte Aggregation
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《动脉硬化血栓血管生物学》
From the Division of Cardiology (M.M., T.M.) and the Department of Internal Medicine and Department of Transfusion Medicine (K.G., P.K.), University Hospital Hamburg-Eppendorf, Hamburg, Germany; the Division of Cardiology (C.B.), Department of Internal Medicine, University Hospital Rostock, Germany; and the Department of Pathology and Medicine (P.T.), Baylor College of Medicine, Houston, Tex.
Correspondence to Dr Michael Merten, Division of Cardiology, Department of Internal Medicine, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. E-mail michael_merten@yahoo.com
Abstract
Objective— Sulfatides are sulfated glycosphingolipids present on the surface of a variety of cells; however, their exact physiological function is not known. Recently, we have shown that the inhibition of sulfatide–P-selectin interactions leads to disaggregation of platelet aggregates.
Methods and Results— In this study, we show that sulfatides activated platelets as they increased activation of GPIIb/IIIa (PAC-1 epitope) and expression of P-selectin on the platelet surface. Furthermore, sulfatides aggregated washed platelets in a dose-dependent manner and enhanced platelet aggregation in platelet-rich plasma. Previous activation of platelets was necessary for this effect. Monoclonal anti–P-selectin antibodies inhibited not only sulfatide-induced PAC-1 binding to platelets but also sulfatide-induced platelet aggregation, suggesting that sulfatides activate platelet GPIIb/IIIa via signaling through P-selectin. The proaggegatory effect of sulfatides was also observed in an ex vivo thrombosis model using whole blood and pulsatile flow at 37°C. In this model, sulfatides significantly enhanced platelet aggregation and the formation of platelet–leukocyte aggregates.
Conclusions— We show that sulfatide-P-selectin interactions lead to subsequent platelet activation and P-selectin expression, forming a positive feedback loop that can potentiate formation of stable platelet aggregates. In addition, sulfatides enhance the aggregation of platelet–leukocyte aggregates. These mechanisms may play a significant role in hemostasis and thrombosis.
Sulfatides, sulfated glycosphingolipids, activated platelets via P-selectin as determined by an increase in activation of platelet GPIIb/IIIa and P-selectin. Therefore, sulfatides provide a positive feedback loop that can potentiate platelet aggregation. Furthermore, sulfatides aggregated washed platelets in a dose-dependent manner. In an ex vivo thrombosis model, sulfatides enhanced platelet and platelet–leukocyte aggregation.
Key Words: sulfatides ? P-selectin ? platelet aggregation ? platelet–leukocyte aggregation
Introduction
Sulfatides are 3-sulfated galactosyl ceramides expressed on the surface of a variety of cells.1,2 They appear to be involved in cell adhesion2 and nerve conduction.3
Sulfatides interact with several cell adhesion molecules involved in hemostasis such as von Willebrand factor,2 P-selectin,4,5 and thrombospondin.6 Both anticoagulant and procoagulant roles have been proposed for exogenous sulfatides.7 Sulfatides and their analogs are also being developed as candidates for anticoagulant drugs.8 These observations suggest a general role for sulfatides in hemostasis and thrombosis.
After platelet activation, platelet glycoprotein (GP) IIb/IIIa receptor becomes competent to bind soluble fibrinogen, which bridges GPIIb/IIIa between adjacent platelets.9 As activation progresses, platelets secrete their granular contents, and P-selectin, which is present in the granules, translocates to the outer surface.10 P-selectin binds to sulfatides on adjacent already-bridged platelets, and thereby stabilizes platelet aggregates.5 In this study, we provide further insights into the underlying mechanisms by showing that the interaction of sulfatides with platelet P-selectin lead to further platelet activation and further P-selectin expression, thereby providing a positive feedback loop that can potentiate formation of stable platelet aggregates. Furthermore, sulfatides enhanced the formation of platelet–leukocyte aggregates. These mechanisms may play a significant role in hemostasis and thrombosis.
Methods
Antibodies and Reagents
Monoclonal anti–P-selectin antibody G1 was purchased from Biosource International (Camarillo, Calif) and was also a kind gift from Dr Rodger McEver (University of Oklahoma). Monoclonal anti–P-selectin antibody CLB-thromb/6 was obtained from Accurate Chemical & Scientific Corporation, Westbury, NY. Fluorescein isothiocyanate (FITC)-labeled antibody PAC-1, FITC-labeled anti-CD45, PE-labeled anti-CD41a, and PE-labeled anti-CD62P (P-selectin) antibodies were obtained from Becton Dickinson (San Jose, Calif). FITC-labeled goat anti-mouse IgG and IgM were obtained from Sigma (St. Louis, Mo). All antibodies were extensively dialyzed in hepes-buffered saline (0.15 mol/L NaCl, 10 mmol/L hepes, pH 7.5) before their use. Tirofiban was obtained from Merck & Co, Inc. Recombinant annexin V was produced, as previously described.11 Sulfatides, which were obtained from Matreya (Pleasant Gap, Pa) and Avanti (Alabaster, Ala), were chromatographically pure. Phosphatidylcholine was purchased from Sigma. Malaria circumsporozoite protein (MCSP) was produced as described previously, and its binding specificity has been described previously.5
Measurement of Platelet Activation Markers by Flow Cytometry
Washed platelets were prepared by centrifugation of citrated blood from healthy volunteers according to institutional guidelines, as previously described.5,12
Sulfatides (12.5 μg/mL, 25 μg/mL, and 50 μg/mL) or phosphatidylcholine (30 μg/mL), either as micelles or liposomes, were added to washed platelets at 37°C for 5 minutes and fixed with 1% paraformaldehyde. FITC-labeled PAC-1 (20 μg/mL) or PE-labeled anti–P-selectin antibody (20 μg/mL) were added, and fluorescence intensity was measured with a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif) after 45 minutes. In inhibition studies, platelet aliquots were pre-incubated with 10 μg/mL of various antibodies for 5 minutes before the addition of sulfatides (30 μg/mL). As positive controls for PAC-1 binding and P-selectin expression of platelets, 30 μmol/L ADP or 20 μmol/L thrombin receptor activating peptide (TRAP) was used.
To examine whether ADP and sulfatides show additive effects on platelet P-selectin expression, sulfatides (50 μg/mL) were added to washed platelets or platelets in whole blood at 37°C 5 minutes before the addition of ADP (30 μmol/L to washed platelets, 10 μmol/L to whole blood platelets). Subsequently, platelet P-selectin expression was analyzed using a flow cytometer, as described. Whole blood samples were analyzed in the presence of the anti-GPIIb/IIIa antagonist tirofiban (50 μg/mL). Tirofiban had only a minimal inhibitory effect (< 8% inhibition) on P-selectin expression induced by ADP and inhibited the formation of platelet aggregates in whole blood almost completely, allowing the measurement of P-selectin expression on a homogenous population of single platelets.13
Sulfatide Micelles and Liposomes
Sulfatide micelles were prepared as previously described.5 Sulfatide liposomes were prepared with a molar ratio of sulfatides to cholesterol of 2:1. Sulfatide and cholesterol were dissolved in chloroform, evaporated to dryness under a stream of nitrogen, resuspended in hepes-buffered saline, and emulsified by sonication at 22°C for 45 minutes, as described. Sulfatide concentrations of sulfatide micelles and liposomes were determined as described by Kean.14 Control phosphatidylcholine micelles and liposomes were prepared as described for sulfatides.
Platelet and Platelet–Leukocyte Aggregation Studies in the Aggregometer
Platelets in human platelet-rich plasma (PRP) (2.5x105 platelets/μL) were activated by 3 μmol/L TRAP SFLLRNA or 5 μmol/L ADP, and aggregation was measured in an aggregometer (Biodata, Horsham, Pa).5,12
The effect of antibody CLB-thromb/6 (10 μg/mL), antibody G1 (10 μg/mL), recombinant MCSP (20 μg/mL), antibody WM23 (40 μg/mL), control antibody (40 μg/mL), or recombinant annexin V (20 μg/mL) was determined by pre-incubation at 37°C for 5 minutes before the addition of agonists. To examine the effect of sulfatides on platelets in plasma, PRP was incubated with 50 μg/mL sulfatides, either as micelles or liposomes, at 37° for 5 minutes before the addition of agonists (TRAP, ADP) in the aggregometer. Phosphatidylcholine was used as control.
To further determine the effect of sulfatides on washed platelets, various concentrations of sulfatides ranging from 15 to 45 μg/mL were added to platelets at 37°C for 5 minutes before stirring in the aggregometer. The effect of monoclonal antibodies or recombinant proteins was determined by pre-incubation at 37°C for 5 minutes before the addition of sulfatides.
The effects of sulfatide antagonist MCSP on sulfatide-induced platelet and platelet–leukocyte aggregation in whole blood were measured in the aggregometer. Sulfatides (50 μg/mL) were added to citrated whole blood; after 5 minutes of incubation, ADP (3 μmol/L) was added, and aliquots were taken at intervals of 0, 5, 10, and 15 minutes. The sulfatide antagonist MCSP (20 μg/mL) or a control protein (phosphatidylserine binding annexin V, 20 μg/mL) was added 3 minutes before the addition of ADP to examine its effect on platelet and platelet–leukocyte aggregation. To measure platelet aggregation, the 50-μL whole blood aliquots were fixed with 1% paraformaldehyde, and 20 μL FITC-labeled anti-CD42b antibody was added. After 30 minutes of incubation, the samples were diluted and analyzed in a flow cytometer. Unactivated whole blood samples were used to set a gate for events representing CD42b-positive single platelets. With these settings, the disappearance of the single platelet population (in the preset gate) is a measure of the degree of platelet aggregation, as previously described.13
For the measurement of platelet leukocyte aggregation, the whole blood aliquots were fixed, and 10 μL PE-labeled anti-CD41a antibody and 10 μL FITC-labeled anti-CD45 antibody were added. After 30 minutes of incubation, the samples were analyzed in a flow cytometer. The forward and sideward scatter and PE fluorescence profiles were acquired on a logarithmic scale, whereas FITC fluorescence intensity was detected with a linear detection setting. Leukocytes were distinguished from other cells using forward and sideward scatter, as well as a fluorescence threshold set to detect only those cells positive for leukocyte-specific marker anti–CD45-FITC. Leukocyte–platelet aggregates were considered those particles identified as leukocytes that expressed platelet marker anti-CD41a–PE fluorescence above a background level. The percentage of leukocytes binding platelets was then expressed as the percentage of leukocytes with positive platelet marker above the threshold. Isotype-matched control antibodies were used to set thresholds.
Measurement of Platelet Aggregation and Platelet–Leukocyte Conjugate Formation in an Ex Vivo Thrombosis Model
A previously described and modified ex vivo thrombosis model15 was used to examine platelet and platelet–leukocyte aggregation under more physiological conditions. An 80-cm silicon tubing with an inner diameter of 3 mm was carefully filled with 6 mL citrated whole blood via a 3-way faucet and placed in a water bath at 37°C. The whole blood in the tubing was rotated by a roller pump (Ismatec, Zurich, Switzerland) at a rotation rate effecting 100 pulsations per minute and a flow rate of 10 mL/min. Sulfatide micelles (75 μg/mL) or control phosphatidylcholine micelles (75 μg/mL) were added via the 3-way faucet after the tubing had been filled with whole blood and incubated at 37°C for 3 minutes. The roller pump was started, and after 1 rotation the first aliquot was taken (0 minute). Subsequently, ADP (3 μmol/L) was added, and additional aliquots were taken at intervals of 3, 6, 12, 20, and 30 minutes via the 3-way faucet from the thrombosis model. To measure platelet and platelet–leukocyte aggregation, whole blood aliquots were fixed and analyzed in a flow cytometer, as described.
Statistical Analysis
All experimental values are represented as mean±SD. Statistical significance was evaluated by 1-way ANOVA followed by the Tukey–Kramer multiple comparisons test. P<0.05 was considered statistically significant.
Results
Stimulatory Effect of Sulfatides on Platelets
Sulfatides, either as micelles or cholesterol-sulfatide liposomes, induced a concentration-dependent increase of PAC-1 binding to platelets, whereas phosphatidylcholine micelles or liposomes had no effect (n=3, P<0.001) (Figure 1A and 1B). Sulfatide micelles in a concentration of 50 μg/mL induced a maximal increase of PAC-1 binding to platelets, which was 17-fold greater than that of the control. Beyond a concentration of 50 μg/mL, there was no further increase in PAC-1 binding to platelets (data not shown). The increase of PAC-1 binding induced by sulfatides was similar to the one induced by 20 μmol/L thrombin receptor-activating peptide, and 60% stronger than the one induced by 30 μmol/L ADP (Figure 1A and 1B). The monoclonal anti–P-selectin antibodies, CLB-thromb/6 and G1, inhibited the sulfatide-induced PAC-1 binding to platelets by 87% and 60%, respectively (n=3, P<0.001) (Figure 1C), suggesting that sulfatides activate platelet GPIIb/IIIa via signaling through P-selectin. The different inhibitory effects of the anti–P-selectin antibodies, CLB-thromb/6 and G1, may be explained by different epitopes of the antibodies within the functional lectin domain of P-selectin. The control antibody had no effect on sulfatide-induced PAC-1 expression on platelets.
Figure 1. Effect of sulfatides on PAC-1 binding to platelets. A, Sulfatides in different concentrations (12.5 μg/mL, 25 μg/mL, and 50 μg/mL) or phosphatidylcholine micelles (30 μg/mL) were added to washed platelets, followed by FITC-labeled PAC-1. The fluorescence associated with platelets was determined by flow cytometry. For comparison, the platelet agonists ADP (30 μmol/L) and TRAP (20 μmol/L) were added. Sulfatide micelles in a concentration of 50 μg/mL induced a maximal increase of PAC-1 binding to platelets, which was 17-fold greater than that of the control (n=3, P<0.001). B, Representative immunofluorescence histogram shows PAC-1 binding to platelets in presence of control phosphatidylcholine (white), sulfatides (50 μg/mL) (black), or TRAP (20 μmol/L) (gray). C, For inhibition studies, platelets were pre-incubated with 10 μg/mL of control antibody (control), or anti–P-selectin antibodies, CLB-thromb/6 and G1, for 5 minutes before the addition of sulfatides (30 μg/mL). The monoclonal anti–P-selectin antibodies, CLB-thromb/6 and G1, inhibited the sulfatide-induced PAC-1 binding to platelets by 87% and 60%, respectively (n=3, P<0.001).
Platelets, washed by centrifugation steps, expressed significantly more P-selectin than PRP platelets (Figure 2) (n=3, P<0.001) with a similar degree of PAC-1 binding (data not shown), despite having prostaglandin E1 in the washing buffers, and this P-selectin mediates the proaggregatory effect of sulfatides. Because washed platelets had higher expression of P-selectin on their surface, sulfatides can better interact with washed platelets via P-selectin than with PRP platelets. Thus, sulfatides increased the expression of P-selectin on washed platelets in a concentration-dependent manner with a maximal increase of 88% at a concentration of 50 μg/mL (n=3, P<0.001), whereas phosphatidylcholine did not induce P-selectin expression (Figure 2). This increase in P-selectin expression induced by sulfatides was comparable to the effect of 30 μmol/L ADP and 40% of the effect of 20 μmol/L TRAP (Figure 2). Compared with TRAP, sulfatides are not such a strong agonist and therefore do not have a similar effect on P-selectin expression. During platelet activation, a different threshold of stimulation exists for aggregation and alpha granule release. Alpha granule release has a higher threshold of activation than the conformational changes of the GP IIb/IIIa complex. Concentrations of ADP, which induce 100% fibrinogen binding, cause only 33% P-selectin expression.16
Figure 2. Effect of sulfatides on platelet P-selectin expression. Sulfatides in different concentrations (12.5 μg/mL, 25 μg/mL, and 50 μg/mL) or phosphatidylcholine micelles (PC; 30 μg/mL) were added to washed platelets, followed by PE-labeled anti–P-selectin antibody. For comparison, the platelet agonists ADP (30 μmol/L) and TRAP (20 μmol/L) were added. The fluorescence associated with platelets was determined by flow cytometry. Sulfatides increased the expression of P-selectin on washed platelets in a concentration-dependent manner with a maximal increase of 88% at a concentration of 50 μg/mL (n=3, P<0.001). B, Representative immunofluorescence histogram shows platelet P-selectin expression in presence of control phosphatidylcholine (white), sulfatides (50 μg/mL) (black), or TRAP (20 μmol/L) (gray).
Proaggegatory Effect of Sulfatides on Platelet Aggregation
Sulfatides themselves had a minimal effect on unactivated platelets in PRP. However, sulfatides showed a proaggregatory effect on activated platelets in PRP, because they increased the extent of platelet aggregation induced by TRAP by 23±5% compared with the phosphatidylcholine control (n=5, P<0.01) (Figure 3A). Similar proaggregatory effects of sulfatides were seen after activation of platelets with 5 μmol/L ADP (data not shown). The concentration of sulfatides that had a maximal proaggregatory effect was 50 μg/mL.
Figure 3. Effect of sulfatides on platelet aggregation using the aggregometer. A, Platelet aggregation in PRP incubated for 5 minutes with either phosphatidylcholine micelles (1 and 3) or sulfatide micelles (each 50 μg/mL) (2 and 4) was measured in an aggregometer. TRAP (3 μmol/L) was included for samples 3 and 4. Sulfatides increased the extent of platelet aggregation induced by TRAP by 23±5% compared with the phosphatidylcholine control (n=5, P<0.001). Results shown are representative of 5 separate experiments. B, Aggregation of washed platelets incubated with control phosphatidylcholine micelles (45 μg/mL) (1), or sulfatide micelles at 15 μg/mL (2), 30 μg/mL (3), or 45 μg/mL (4) were measured as mentioned. Results shown are representative of 5 separate experiments. C, Aggregation of washed platelets incubated with sulfatide micelles (30 μg/mL) in the presence of control monoclonal antibody (20 μg/mL) (1), recombinant annexin V (20 μg/mL) (2), monoclonal anti-GPIb antibody WM23 (3), monoclonal anti–P-selectin antibody CLB-thromb/6 (10 μg/mL) (4), or recombinant malaria circumsporozoite protein (20 μg/mL) (5). Results shown are representative of 3 separate experiments.
In washed platelets, sulfatides induced significant platelet aggregation in a dose-dependent and saturable manner, peaking near at 45 μg/mL, whereas phosphatidylcholine had no effect (Figure 3B). The proaggregatory effect of sulfatides was very much more pronounced in washed platelets than in PRP (Figure 3A and 3B), consistent with the significantly higher level of surface P-selectin in washed platelets than in PRP platelets (Figure 2). The monoclonal anti–P-selectin antibody, CLB-thromb/6, inhibited sulfatide-induced platelet aggregation by 80% to 90%, whereas anti-GPIb antibody WM23 and isotype-matched control antibody had no effect (Figure 3C). Also, sulfatide-binding recombinant malaria circumsporozoite protein5 inhibited sulfatide-induced platelet aggregation by 80% to 90%, whereas phosphatidylserine-binding annexin V had no effect (Figure 3C). These results indicate that the interaction of sulfatides with P-selectin can trigger aggregation of washed platelets.
Effect of Sulfatides on P-Selectin Expression of Platelets Activated With ADP
We investigated whether sulfatides only exert an activating effect on already-activated platelets (because these platelets express P-selectin) by examining the effect of sulfatides on platelet P-selectin expression with and without activation with ADP. Sulfatides themselves did not increase P-selectin expression on unactivated platelets in whole blood, but they did increase P-selectin expression on washed platelets, which are partially activated because of isolation procedures (Figure I, available online at http://atvb.ahajournals.org). However, after activation with ADP, sulfatides increased the expression of P-selectin in washed platelets by 135%, and in whole blood platelets by 39% (n=3, P<0.001, respectively), demonstrating that previous activation of platelets by isolation procedures or ADP (with subsequent P-selectin expression) is necessary for sulfatides to further activate platelets (Figure I). The activating effect of sulfatides on activated platelets in whole blood is much weaker than the effect on activated washed platelets, probably because whole blood contains a number of sulfatide-binding proteins that diminish the effect of exogenous sulfatides. The weak agonist ADP induced only minimal activation in washed platelets compared with whole blood platelets, probably because of some remaining effects of prostaglandin E1 in washed platelets.
Sulfatides Enhance Platelet Aggregation in an Ex Vivo Thrombosis Model
To investigate the effect of sulfatides on platelet aggregation under more physiological conditions, we used an ex vivo thrombosis model, in which human citrated whole blood was filled in a circular silicon tubing with a diameter similar to coronary arteries, and pulsatile flow with a velocity of 10 mL/min was used at 37°C. In this model, we used ADP as platelet agonist in a low concentration (3 μmol/L) to better demonstrate the stabilizing effect of sulfatides on platelet aggregation. At this ADP concentration, not all platelets aggregate irreversibly; therefore, platelet disaggregation occurs over time. However, sulfatide micelles (75 μg/mL) significantly reduced this disaggregation and enhanced the formation of stable platelet aggregates (Figure 4A). Under similar conditions, control phosphatidylcholine micelles had no enhancing effect on platelet aggregation. Sulfatide concentrations had to be higher in the ex vivo thrombosis model than in platelet aggregation experiments, probably because of to a significant absorption of sulfatides to the walls of the tubing in our thrombosis model. At 30 minutes after initial platelet activation, the platelet aggregate stabilizing effect of sulfatide micelles led to an increase in platelet aggregation by 40% (Figure 4A). Using hirudin-anticoagulated whole blood, sulfatide micelles stabilized platelet aggregates to a similar degree than with citrate-anticoagulated blood (data not shown), suggesting that the sulfatide effect on platelets is independent of the calcium concentration. However, sulfatide micelles had no significant effect on platelets not previously activated with ADP in the ex vivo thrombosis model, suggesting a secondary role of sulfatides in platelet aggregation after initial platelet activation. These data are consistent with the finding that sulfatides increased platelet P-selectin expression only in platelets that were previously activated.
Figure 4. Effect of sulfatides on platelet aggregation in whole blood using an ex vivo thrombosis model. A, Sulfatides (75 μg/mL) or control phosphatidylcholine micelles (75 μg/mL) were added to whole blood, incubated at 37°C for 3 minutes, and activated with 3 μmol/L ADP. A circular silicon tubing was filled with the blood and pulsatile flow of 10 mL/min was used at 37°C. Aliquots of blood taken at different time intervals and platelet aggregation were measured by flow cytometry. Sulfatide micelles (?), phosphatidylcholine micelles (), phosphate-buffered saline (PBS) (), sulfatide micelles without ADP (), and phosphatidylcholine micelles without ADP (). B, Aggregation of whole blood platelets after addition of sulfatides (50 μg/mL) and 3 μmol/L ADP in presence of either control (annexin V, 20 μg/mL) or MCSP (20 μg/mL) in the aggregometer is shown. Aliquots of blood were taken at different time intervals, and platelet aggregation was measured by flow cytometry. Control annexin V (?), MCSP ().
To confirm the role of sulfatides in platelet aggregation in whole blood, we examined the effect of the sulfatide antagonist MCSP5 on sulfatide-induced platelet aggregation. MCSP inhibited sulfatide-induced platelet aggregation in whole blood by 39% at 15 minutes after initial platelet activation (Figure 4B).
Sulfatides Enhance the Formation of Platelet–Leukocyte Conjugates in an Ex Vivo Thrombosis Model
We also examined the influence of sulfatide micelles on the formation of platelet–leukocyte aggregates in whole blood using the ex vivo thrombosis model. Sulfatides (75 μg/mL) enhanced the formation of platelet–leukocyte aggregates induced by the addition of 3 μmol/L ADP, whereas control phosphatidylcholine micelles had no enhancing effect on platelet–leukocyte aggregation (Figure 5A and 5B). The formation of platelet–leukocyte aggregates induced by ADP increased progressively over time, as also shown by Konstantopoulos et al for shear-induced platelet–leukocyte aggregation.17 At 30 minutes after initial activation with ADP, sulfatide micelles enhanced the formation of platelet–leukocyte aggregate by 70% (Figure 5A). The addition of sulfatide micelles to whole blood not previously activated by ADP had no significant enhancing effect, suggesting that an activated state of the platelets is necessary for sulfatides to enhance the formation of platelet–leukocyte aggregates. To confirm a role of sulfatides in the formation of platelet–leukocyte aggregates, we examined the effect of sulfatide antagonist MCSP on sulfatide-induced platelet–leukocyte aggregation. MCSP inhibited the sulfatide-induced formation of platelet–leukocyte aggregates by 62% at 15 minutes after the addition of ADP (Figure 5C).
Figure 5. Effect of sulfatides on platelet–leukocyte aggregation in whole blood using an ex vivo thrombosis model. A, Conditions were similar to those described in Figure 4, and platelet–leukocyte aggregation in presence of either phosphatidylcholine or sulfatide micelles was measured after addition of 3 μmol/L ADP using flow cytometry. Sulfatide micelles (?), phosphatidylcholine micelles (), PBS (), sulfatide micelles without ADP (), and phosphatidylcholine micelles without ADP (). B, Representative immunofluorescence histogram shows leukocytes that expressed platelet marker anti-CD41a–PE fluorescence above a background level (101 fluorescence) at 30 minutes after addition of buffer (white), ADP with phosphatidylcholine (75 μg/mL) (gray), or ADP with sulfatides (75 μg/mL) (black). C, Platelet–leukocyte aggregation in citrated whole blood after addition of sulfatides (50 μg/mL) and 3 μmol/L ADP in the presence of either control (annexin V, 20 μg/mL) or MCSP (20 μg/mL) in the aggregometer is shown. Aliquots of whole blood were taken at different time intervals, and platelet–leukocyte aggregation was measured by flow cytometry. Control annexin V (?), MCSP ().
Discussion
In this study, we show that the interaction between sulfatides and P-selectin leads to further platelet activation, as determined by the increase in surface expression of P-selectin and activated GPIIb/IIIa (PAC-1 epitope) on platelets, and the formation of platelet and platelet–leukocyte aggregates. Monoclonal anti–P-selectin antibodies inhibited these effects, suggesting a signal-transducing role for P-selectin in platelets similar to the one recently described for L-selectin in neutrophils.18 The platelet activating effect of sulfatides provides a positive feedback loop that potentiates formation of stable platelet aggregates and platelet–leukocyte aggregates. This amplification mechanism may be important for the formation of stable platelet thrombi at sites of vascular injuries, especially in the arterial circulation. The addition of sulfatides to unstimulated blood had no significant effect, suggesting previous activation of platelets (with P-selectin expression) is necessary for sulfatides to exhibit their activating effects. A similar activation-dependent proaggregatory effect of sulfatides was seen in platelet and platelet–leukocyte aggregation under more physiological conditions of flowing blood in an ex vivo thrombosis model. Unlike the P-selectin expression in platelets, the expression of the leukocyte ligand for sulfatides, L-selectin, is independent of its activation state. Our finding that previous activation of platelets by ADP is necessary for the proaggregatory effects of sulfatides on platelet and platelet–leukocyte aggregation suggest that sulfatides mediate their effect via platelets and not leukocytes. These proaggregatory effects of sulfatides were inhibited by the sulfatide antagonist MCSP.
The newly described role of sulfatides in platelet–leukocyte aggregation is of importance, because platelet-leukocyte aggregates are recognized to have proinflammatory properties, and increased levels are associated with an increased risk for the formation of arterial thrombi. Recent studies showed increased levels of platelet–leukocyte aggregates in patients with unstable angina,19 and after coronary angioplasty.20 Platelet–leukocyte aggregates were also noted to be an early indicator of acute myocardial infarction.20
Recently, we have shown that platelets express sulfatides on their surface, which increase after activation.5 Another study has shown that granulocytes express cell surface sulfatides and excrete sulfatides into medium.4 Thus, both excreted or surface-expressed sulfatides on platelets and granulocytes may enhance platelet and platelet–leukocyte aggregation via P-selectin. Consistent with this, it has been shown that anti–P-selectin antibodies inhibited both platelet–platelet5,12 and platelet–leukocyte aggregation.21
The sulfatide–P-selectin interactions may also be important in the adhesion of platelets to sulfatides on certain cancer cells5 or in atherosclerotic lesions,22 both of which are associated with prothrombotic conditions. The cell surface sulfatides may provide a nidus for adhesion and activation of both platelets and leukocytes, thereby contributing to the pathophysiological mechanisms of the coronary syndrome.
Sulfatide and sialyl lewis x analogs have been used in vivo to block selectin functions. In animal models, these compounds inhibit P-selectin–dependent organ injuries.23 Inhibition of P-selectin function also had antithrombotic effects in many studies. P-selectin inhibition not only accelerated thrombolysis in a primate model of arterial thrombosis24 but also reduced the extent of venous thrombosis in another primate model25 and in recurrent coronary arterial thrombosis in dogs.26 The observed beneficial effects of P-selectin blockade may be caused by the inhibition of sulfatide–P-selectin interactions in platelet and platelet–leukocyte aggregation. However, so far no clinical studies have been reported examining the effect of P-selectin inhibition on hemostasis and thrombosis.
In summary, we show that sulfatides activate platelets via P-selectin and enhance platelet and platelet–leukocyte aggregation. This mechanism may play a significant role in arterial thrombosis, in which platelet and platelet–leukocyte thrombi are formed. Consequently, therapeutic interventions directed against sulfatides or P-selectin may be beneficial in the treatment of arterial thrombosis.
Received April 26, 2004; accepted October 15, 2004.
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Ott I, Neumann FJ, Gawaz M, Schmitt M, Schomig A. increased neutrophil-platelet adhesion in patients with unstable angina. Circulation. 1996; 94: 1239–1246.
Michelson AD, Barnard MR, Krueger LA, Valeri CR, Furman MI. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation. 2001; 104: 1533–1537.
Caron A, Theoret JF, Mousa SA, Merhi Y. Anti-platelet effects of GPIIb/IIIa and P-selectin antagonism, platelet activation, and binding to neutrophils. J Cardiovasc Pharmacol. 2002; 40: 296–306.
Hara A, Taketomi T. Characterization and changes of glycosphingolipids in the aorta of the Watanabe hereditable hyperlipidemic rabbit. J Biochem (Tokyo). 1991; 109: 904–908.
Mulligan MS, Warner RL, Lowe JB, Smith PL, Suzuki Y, Miyasaka M, Yamaguchi S, Ohta Y, Tsukada Y, Kiso M, Hasegawa A, Ward PA. In vitro and in vivo selectin-blocking activities of sulfated lipids and sulfated sialyl compounds. Int Immunol. 1998; 10: 569–575.
Toombs CF, DeGraaf GL, Martin JP, Geng JG, Anderson DC, Shebuski RJ. Pretreatment with a blocking monoclonal antibody to P-selectin accelerates pharmacological thrombolysis in a primate model of arterial thrombosis. J Pharmacol Exp Ther. 1995; 275: 941–949.
Downing LJ, Wakefield TW, Strieter RM, Prince MR, Londy FJ, Fowlkes JB, Hulin MS, Kadell AM, Wilke CA, Brown SL, Wrobleski SK, Burdick MD, Anderson DC, Greenfield LJ. Anti–P-selectin antibody decreases inflammation and thrombus formation in venous thrombosis. J Vasc Surg. 1997; 25: 816–828.
Ikeda H, Ueyama T, Murohara T, Yasukawa H, Haramaki N, Eguchi H, Katoh A, Takajo Y, Onitsuka I, Ueno T, Tojo SJ, Imaizumi T. Adhesive interaction between P-selectin and sialyl Lewis(x) plays an important role in recurrent coronary arterial thrombosis in dogs. Arterioscler Thromb Vasc Biol. 1999; 19: 1083–1090.(Michael Merten; Christian)
Correspondence to Dr Michael Merten, Division of Cardiology, Department of Internal Medicine, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. E-mail michael_merten@yahoo.com
Abstract
Objective— Sulfatides are sulfated glycosphingolipids present on the surface of a variety of cells; however, their exact physiological function is not known. Recently, we have shown that the inhibition of sulfatide–P-selectin interactions leads to disaggregation of platelet aggregates.
Methods and Results— In this study, we show that sulfatides activated platelets as they increased activation of GPIIb/IIIa (PAC-1 epitope) and expression of P-selectin on the platelet surface. Furthermore, sulfatides aggregated washed platelets in a dose-dependent manner and enhanced platelet aggregation in platelet-rich plasma. Previous activation of platelets was necessary for this effect. Monoclonal anti–P-selectin antibodies inhibited not only sulfatide-induced PAC-1 binding to platelets but also sulfatide-induced platelet aggregation, suggesting that sulfatides activate platelet GPIIb/IIIa via signaling through P-selectin. The proaggegatory effect of sulfatides was also observed in an ex vivo thrombosis model using whole blood and pulsatile flow at 37°C. In this model, sulfatides significantly enhanced platelet aggregation and the formation of platelet–leukocyte aggregates.
Conclusions— We show that sulfatide-P-selectin interactions lead to subsequent platelet activation and P-selectin expression, forming a positive feedback loop that can potentiate formation of stable platelet aggregates. In addition, sulfatides enhance the aggregation of platelet–leukocyte aggregates. These mechanisms may play a significant role in hemostasis and thrombosis.
Sulfatides, sulfated glycosphingolipids, activated platelets via P-selectin as determined by an increase in activation of platelet GPIIb/IIIa and P-selectin. Therefore, sulfatides provide a positive feedback loop that can potentiate platelet aggregation. Furthermore, sulfatides aggregated washed platelets in a dose-dependent manner. In an ex vivo thrombosis model, sulfatides enhanced platelet and platelet–leukocyte aggregation.
Key Words: sulfatides ? P-selectin ? platelet aggregation ? platelet–leukocyte aggregation
Introduction
Sulfatides are 3-sulfated galactosyl ceramides expressed on the surface of a variety of cells.1,2 They appear to be involved in cell adhesion2 and nerve conduction.3
Sulfatides interact with several cell adhesion molecules involved in hemostasis such as von Willebrand factor,2 P-selectin,4,5 and thrombospondin.6 Both anticoagulant and procoagulant roles have been proposed for exogenous sulfatides.7 Sulfatides and their analogs are also being developed as candidates for anticoagulant drugs.8 These observations suggest a general role for sulfatides in hemostasis and thrombosis.
After platelet activation, platelet glycoprotein (GP) IIb/IIIa receptor becomes competent to bind soluble fibrinogen, which bridges GPIIb/IIIa between adjacent platelets.9 As activation progresses, platelets secrete their granular contents, and P-selectin, which is present in the granules, translocates to the outer surface.10 P-selectin binds to sulfatides on adjacent already-bridged platelets, and thereby stabilizes platelet aggregates.5 In this study, we provide further insights into the underlying mechanisms by showing that the interaction of sulfatides with platelet P-selectin lead to further platelet activation and further P-selectin expression, thereby providing a positive feedback loop that can potentiate formation of stable platelet aggregates. Furthermore, sulfatides enhanced the formation of platelet–leukocyte aggregates. These mechanisms may play a significant role in hemostasis and thrombosis.
Methods
Antibodies and Reagents
Monoclonal anti–P-selectin antibody G1 was purchased from Biosource International (Camarillo, Calif) and was also a kind gift from Dr Rodger McEver (University of Oklahoma). Monoclonal anti–P-selectin antibody CLB-thromb/6 was obtained from Accurate Chemical & Scientific Corporation, Westbury, NY. Fluorescein isothiocyanate (FITC)-labeled antibody PAC-1, FITC-labeled anti-CD45, PE-labeled anti-CD41a, and PE-labeled anti-CD62P (P-selectin) antibodies were obtained from Becton Dickinson (San Jose, Calif). FITC-labeled goat anti-mouse IgG and IgM were obtained from Sigma (St. Louis, Mo). All antibodies were extensively dialyzed in hepes-buffered saline (0.15 mol/L NaCl, 10 mmol/L hepes, pH 7.5) before their use. Tirofiban was obtained from Merck & Co, Inc. Recombinant annexin V was produced, as previously described.11 Sulfatides, which were obtained from Matreya (Pleasant Gap, Pa) and Avanti (Alabaster, Ala), were chromatographically pure. Phosphatidylcholine was purchased from Sigma. Malaria circumsporozoite protein (MCSP) was produced as described previously, and its binding specificity has been described previously.5
Measurement of Platelet Activation Markers by Flow Cytometry
Washed platelets were prepared by centrifugation of citrated blood from healthy volunteers according to institutional guidelines, as previously described.5,12
Sulfatides (12.5 μg/mL, 25 μg/mL, and 50 μg/mL) or phosphatidylcholine (30 μg/mL), either as micelles or liposomes, were added to washed platelets at 37°C for 5 minutes and fixed with 1% paraformaldehyde. FITC-labeled PAC-1 (20 μg/mL) or PE-labeled anti–P-selectin antibody (20 μg/mL) were added, and fluorescence intensity was measured with a FACScan flow cytometer (Becton Dickinson, Mountain View, Calif) after 45 minutes. In inhibition studies, platelet aliquots were pre-incubated with 10 μg/mL of various antibodies for 5 minutes before the addition of sulfatides (30 μg/mL). As positive controls for PAC-1 binding and P-selectin expression of platelets, 30 μmol/L ADP or 20 μmol/L thrombin receptor activating peptide (TRAP) was used.
To examine whether ADP and sulfatides show additive effects on platelet P-selectin expression, sulfatides (50 μg/mL) were added to washed platelets or platelets in whole blood at 37°C 5 minutes before the addition of ADP (30 μmol/L to washed platelets, 10 μmol/L to whole blood platelets). Subsequently, platelet P-selectin expression was analyzed using a flow cytometer, as described. Whole blood samples were analyzed in the presence of the anti-GPIIb/IIIa antagonist tirofiban (50 μg/mL). Tirofiban had only a minimal inhibitory effect (< 8% inhibition) on P-selectin expression induced by ADP and inhibited the formation of platelet aggregates in whole blood almost completely, allowing the measurement of P-selectin expression on a homogenous population of single platelets.13
Sulfatide Micelles and Liposomes
Sulfatide micelles were prepared as previously described.5 Sulfatide liposomes were prepared with a molar ratio of sulfatides to cholesterol of 2:1. Sulfatide and cholesterol were dissolved in chloroform, evaporated to dryness under a stream of nitrogen, resuspended in hepes-buffered saline, and emulsified by sonication at 22°C for 45 minutes, as described. Sulfatide concentrations of sulfatide micelles and liposomes were determined as described by Kean.14 Control phosphatidylcholine micelles and liposomes were prepared as described for sulfatides.
Platelet and Platelet–Leukocyte Aggregation Studies in the Aggregometer
Platelets in human platelet-rich plasma (PRP) (2.5x105 platelets/μL) were activated by 3 μmol/L TRAP SFLLRNA or 5 μmol/L ADP, and aggregation was measured in an aggregometer (Biodata, Horsham, Pa).5,12
The effect of antibody CLB-thromb/6 (10 μg/mL), antibody G1 (10 μg/mL), recombinant MCSP (20 μg/mL), antibody WM23 (40 μg/mL), control antibody (40 μg/mL), or recombinant annexin V (20 μg/mL) was determined by pre-incubation at 37°C for 5 minutes before the addition of agonists. To examine the effect of sulfatides on platelets in plasma, PRP was incubated with 50 μg/mL sulfatides, either as micelles or liposomes, at 37° for 5 minutes before the addition of agonists (TRAP, ADP) in the aggregometer. Phosphatidylcholine was used as control.
To further determine the effect of sulfatides on washed platelets, various concentrations of sulfatides ranging from 15 to 45 μg/mL were added to platelets at 37°C for 5 minutes before stirring in the aggregometer. The effect of monoclonal antibodies or recombinant proteins was determined by pre-incubation at 37°C for 5 minutes before the addition of sulfatides.
The effects of sulfatide antagonist MCSP on sulfatide-induced platelet and platelet–leukocyte aggregation in whole blood were measured in the aggregometer. Sulfatides (50 μg/mL) were added to citrated whole blood; after 5 minutes of incubation, ADP (3 μmol/L) was added, and aliquots were taken at intervals of 0, 5, 10, and 15 minutes. The sulfatide antagonist MCSP (20 μg/mL) or a control protein (phosphatidylserine binding annexin V, 20 μg/mL) was added 3 minutes before the addition of ADP to examine its effect on platelet and platelet–leukocyte aggregation. To measure platelet aggregation, the 50-μL whole blood aliquots were fixed with 1% paraformaldehyde, and 20 μL FITC-labeled anti-CD42b antibody was added. After 30 minutes of incubation, the samples were diluted and analyzed in a flow cytometer. Unactivated whole blood samples were used to set a gate for events representing CD42b-positive single platelets. With these settings, the disappearance of the single platelet population (in the preset gate) is a measure of the degree of platelet aggregation, as previously described.13
For the measurement of platelet leukocyte aggregation, the whole blood aliquots were fixed, and 10 μL PE-labeled anti-CD41a antibody and 10 μL FITC-labeled anti-CD45 antibody were added. After 30 minutes of incubation, the samples were analyzed in a flow cytometer. The forward and sideward scatter and PE fluorescence profiles were acquired on a logarithmic scale, whereas FITC fluorescence intensity was detected with a linear detection setting. Leukocytes were distinguished from other cells using forward and sideward scatter, as well as a fluorescence threshold set to detect only those cells positive for leukocyte-specific marker anti–CD45-FITC. Leukocyte–platelet aggregates were considered those particles identified as leukocytes that expressed platelet marker anti-CD41a–PE fluorescence above a background level. The percentage of leukocytes binding platelets was then expressed as the percentage of leukocytes with positive platelet marker above the threshold. Isotype-matched control antibodies were used to set thresholds.
Measurement of Platelet Aggregation and Platelet–Leukocyte Conjugate Formation in an Ex Vivo Thrombosis Model
A previously described and modified ex vivo thrombosis model15 was used to examine platelet and platelet–leukocyte aggregation under more physiological conditions. An 80-cm silicon tubing with an inner diameter of 3 mm was carefully filled with 6 mL citrated whole blood via a 3-way faucet and placed in a water bath at 37°C. The whole blood in the tubing was rotated by a roller pump (Ismatec, Zurich, Switzerland) at a rotation rate effecting 100 pulsations per minute and a flow rate of 10 mL/min. Sulfatide micelles (75 μg/mL) or control phosphatidylcholine micelles (75 μg/mL) were added via the 3-way faucet after the tubing had been filled with whole blood and incubated at 37°C for 3 minutes. The roller pump was started, and after 1 rotation the first aliquot was taken (0 minute). Subsequently, ADP (3 μmol/L) was added, and additional aliquots were taken at intervals of 3, 6, 12, 20, and 30 minutes via the 3-way faucet from the thrombosis model. To measure platelet and platelet–leukocyte aggregation, whole blood aliquots were fixed and analyzed in a flow cytometer, as described.
Statistical Analysis
All experimental values are represented as mean±SD. Statistical significance was evaluated by 1-way ANOVA followed by the Tukey–Kramer multiple comparisons test. P<0.05 was considered statistically significant.
Results
Stimulatory Effect of Sulfatides on Platelets
Sulfatides, either as micelles or cholesterol-sulfatide liposomes, induced a concentration-dependent increase of PAC-1 binding to platelets, whereas phosphatidylcholine micelles or liposomes had no effect (n=3, P<0.001) (Figure 1A and 1B). Sulfatide micelles in a concentration of 50 μg/mL induced a maximal increase of PAC-1 binding to platelets, which was 17-fold greater than that of the control. Beyond a concentration of 50 μg/mL, there was no further increase in PAC-1 binding to platelets (data not shown). The increase of PAC-1 binding induced by sulfatides was similar to the one induced by 20 μmol/L thrombin receptor-activating peptide, and 60% stronger than the one induced by 30 μmol/L ADP (Figure 1A and 1B). The monoclonal anti–P-selectin antibodies, CLB-thromb/6 and G1, inhibited the sulfatide-induced PAC-1 binding to platelets by 87% and 60%, respectively (n=3, P<0.001) (Figure 1C), suggesting that sulfatides activate platelet GPIIb/IIIa via signaling through P-selectin. The different inhibitory effects of the anti–P-selectin antibodies, CLB-thromb/6 and G1, may be explained by different epitopes of the antibodies within the functional lectin domain of P-selectin. The control antibody had no effect on sulfatide-induced PAC-1 expression on platelets.
Figure 1. Effect of sulfatides on PAC-1 binding to platelets. A, Sulfatides in different concentrations (12.5 μg/mL, 25 μg/mL, and 50 μg/mL) or phosphatidylcholine micelles (30 μg/mL) were added to washed platelets, followed by FITC-labeled PAC-1. The fluorescence associated with platelets was determined by flow cytometry. For comparison, the platelet agonists ADP (30 μmol/L) and TRAP (20 μmol/L) were added. Sulfatide micelles in a concentration of 50 μg/mL induced a maximal increase of PAC-1 binding to platelets, which was 17-fold greater than that of the control (n=3, P<0.001). B, Representative immunofluorescence histogram shows PAC-1 binding to platelets in presence of control phosphatidylcholine (white), sulfatides (50 μg/mL) (black), or TRAP (20 μmol/L) (gray). C, For inhibition studies, platelets were pre-incubated with 10 μg/mL of control antibody (control), or anti–P-selectin antibodies, CLB-thromb/6 and G1, for 5 minutes before the addition of sulfatides (30 μg/mL). The monoclonal anti–P-selectin antibodies, CLB-thromb/6 and G1, inhibited the sulfatide-induced PAC-1 binding to platelets by 87% and 60%, respectively (n=3, P<0.001).
Platelets, washed by centrifugation steps, expressed significantly more P-selectin than PRP platelets (Figure 2) (n=3, P<0.001) with a similar degree of PAC-1 binding (data not shown), despite having prostaglandin E1 in the washing buffers, and this P-selectin mediates the proaggregatory effect of sulfatides. Because washed platelets had higher expression of P-selectin on their surface, sulfatides can better interact with washed platelets via P-selectin than with PRP platelets. Thus, sulfatides increased the expression of P-selectin on washed platelets in a concentration-dependent manner with a maximal increase of 88% at a concentration of 50 μg/mL (n=3, P<0.001), whereas phosphatidylcholine did not induce P-selectin expression (Figure 2). This increase in P-selectin expression induced by sulfatides was comparable to the effect of 30 μmol/L ADP and 40% of the effect of 20 μmol/L TRAP (Figure 2). Compared with TRAP, sulfatides are not such a strong agonist and therefore do not have a similar effect on P-selectin expression. During platelet activation, a different threshold of stimulation exists for aggregation and alpha granule release. Alpha granule release has a higher threshold of activation than the conformational changes of the GP IIb/IIIa complex. Concentrations of ADP, which induce 100% fibrinogen binding, cause only 33% P-selectin expression.16
Figure 2. Effect of sulfatides on platelet P-selectin expression. Sulfatides in different concentrations (12.5 μg/mL, 25 μg/mL, and 50 μg/mL) or phosphatidylcholine micelles (PC; 30 μg/mL) were added to washed platelets, followed by PE-labeled anti–P-selectin antibody. For comparison, the platelet agonists ADP (30 μmol/L) and TRAP (20 μmol/L) were added. The fluorescence associated with platelets was determined by flow cytometry. Sulfatides increased the expression of P-selectin on washed platelets in a concentration-dependent manner with a maximal increase of 88% at a concentration of 50 μg/mL (n=3, P<0.001). B, Representative immunofluorescence histogram shows platelet P-selectin expression in presence of control phosphatidylcholine (white), sulfatides (50 μg/mL) (black), or TRAP (20 μmol/L) (gray).
Proaggegatory Effect of Sulfatides on Platelet Aggregation
Sulfatides themselves had a minimal effect on unactivated platelets in PRP. However, sulfatides showed a proaggregatory effect on activated platelets in PRP, because they increased the extent of platelet aggregation induced by TRAP by 23±5% compared with the phosphatidylcholine control (n=5, P<0.01) (Figure 3A). Similar proaggregatory effects of sulfatides were seen after activation of platelets with 5 μmol/L ADP (data not shown). The concentration of sulfatides that had a maximal proaggregatory effect was 50 μg/mL.
Figure 3. Effect of sulfatides on platelet aggregation using the aggregometer. A, Platelet aggregation in PRP incubated for 5 minutes with either phosphatidylcholine micelles (1 and 3) or sulfatide micelles (each 50 μg/mL) (2 and 4) was measured in an aggregometer. TRAP (3 μmol/L) was included for samples 3 and 4. Sulfatides increased the extent of platelet aggregation induced by TRAP by 23±5% compared with the phosphatidylcholine control (n=5, P<0.001). Results shown are representative of 5 separate experiments. B, Aggregation of washed platelets incubated with control phosphatidylcholine micelles (45 μg/mL) (1), or sulfatide micelles at 15 μg/mL (2), 30 μg/mL (3), or 45 μg/mL (4) were measured as mentioned. Results shown are representative of 5 separate experiments. C, Aggregation of washed platelets incubated with sulfatide micelles (30 μg/mL) in the presence of control monoclonal antibody (20 μg/mL) (1), recombinant annexin V (20 μg/mL) (2), monoclonal anti-GPIb antibody WM23 (3), monoclonal anti–P-selectin antibody CLB-thromb/6 (10 μg/mL) (4), or recombinant malaria circumsporozoite protein (20 μg/mL) (5). Results shown are representative of 3 separate experiments.
In washed platelets, sulfatides induced significant platelet aggregation in a dose-dependent and saturable manner, peaking near at 45 μg/mL, whereas phosphatidylcholine had no effect (Figure 3B). The proaggregatory effect of sulfatides was very much more pronounced in washed platelets than in PRP (Figure 3A and 3B), consistent with the significantly higher level of surface P-selectin in washed platelets than in PRP platelets (Figure 2). The monoclonal anti–P-selectin antibody, CLB-thromb/6, inhibited sulfatide-induced platelet aggregation by 80% to 90%, whereas anti-GPIb antibody WM23 and isotype-matched control antibody had no effect (Figure 3C). Also, sulfatide-binding recombinant malaria circumsporozoite protein5 inhibited sulfatide-induced platelet aggregation by 80% to 90%, whereas phosphatidylserine-binding annexin V had no effect (Figure 3C). These results indicate that the interaction of sulfatides with P-selectin can trigger aggregation of washed platelets.
Effect of Sulfatides on P-Selectin Expression of Platelets Activated With ADP
We investigated whether sulfatides only exert an activating effect on already-activated platelets (because these platelets express P-selectin) by examining the effect of sulfatides on platelet P-selectin expression with and without activation with ADP. Sulfatides themselves did not increase P-selectin expression on unactivated platelets in whole blood, but they did increase P-selectin expression on washed platelets, which are partially activated because of isolation procedures (Figure I, available online at http://atvb.ahajournals.org). However, after activation with ADP, sulfatides increased the expression of P-selectin in washed platelets by 135%, and in whole blood platelets by 39% (n=3, P<0.001, respectively), demonstrating that previous activation of platelets by isolation procedures or ADP (with subsequent P-selectin expression) is necessary for sulfatides to further activate platelets (Figure I). The activating effect of sulfatides on activated platelets in whole blood is much weaker than the effect on activated washed platelets, probably because whole blood contains a number of sulfatide-binding proteins that diminish the effect of exogenous sulfatides. The weak agonist ADP induced only minimal activation in washed platelets compared with whole blood platelets, probably because of some remaining effects of prostaglandin E1 in washed platelets.
Sulfatides Enhance Platelet Aggregation in an Ex Vivo Thrombosis Model
To investigate the effect of sulfatides on platelet aggregation under more physiological conditions, we used an ex vivo thrombosis model, in which human citrated whole blood was filled in a circular silicon tubing with a diameter similar to coronary arteries, and pulsatile flow with a velocity of 10 mL/min was used at 37°C. In this model, we used ADP as platelet agonist in a low concentration (3 μmol/L) to better demonstrate the stabilizing effect of sulfatides on platelet aggregation. At this ADP concentration, not all platelets aggregate irreversibly; therefore, platelet disaggregation occurs over time. However, sulfatide micelles (75 μg/mL) significantly reduced this disaggregation and enhanced the formation of stable platelet aggregates (Figure 4A). Under similar conditions, control phosphatidylcholine micelles had no enhancing effect on platelet aggregation. Sulfatide concentrations had to be higher in the ex vivo thrombosis model than in platelet aggregation experiments, probably because of to a significant absorption of sulfatides to the walls of the tubing in our thrombosis model. At 30 minutes after initial platelet activation, the platelet aggregate stabilizing effect of sulfatide micelles led to an increase in platelet aggregation by 40% (Figure 4A). Using hirudin-anticoagulated whole blood, sulfatide micelles stabilized platelet aggregates to a similar degree than with citrate-anticoagulated blood (data not shown), suggesting that the sulfatide effect on platelets is independent of the calcium concentration. However, sulfatide micelles had no significant effect on platelets not previously activated with ADP in the ex vivo thrombosis model, suggesting a secondary role of sulfatides in platelet aggregation after initial platelet activation. These data are consistent with the finding that sulfatides increased platelet P-selectin expression only in platelets that were previously activated.
Figure 4. Effect of sulfatides on platelet aggregation in whole blood using an ex vivo thrombosis model. A, Sulfatides (75 μg/mL) or control phosphatidylcholine micelles (75 μg/mL) were added to whole blood, incubated at 37°C for 3 minutes, and activated with 3 μmol/L ADP. A circular silicon tubing was filled with the blood and pulsatile flow of 10 mL/min was used at 37°C. Aliquots of blood taken at different time intervals and platelet aggregation were measured by flow cytometry. Sulfatide micelles (?), phosphatidylcholine micelles (), phosphate-buffered saline (PBS) (), sulfatide micelles without ADP (), and phosphatidylcholine micelles without ADP (). B, Aggregation of whole blood platelets after addition of sulfatides (50 μg/mL) and 3 μmol/L ADP in presence of either control (annexin V, 20 μg/mL) or MCSP (20 μg/mL) in the aggregometer is shown. Aliquots of blood were taken at different time intervals, and platelet aggregation was measured by flow cytometry. Control annexin V (?), MCSP ().
To confirm the role of sulfatides in platelet aggregation in whole blood, we examined the effect of the sulfatide antagonist MCSP5 on sulfatide-induced platelet aggregation. MCSP inhibited sulfatide-induced platelet aggregation in whole blood by 39% at 15 minutes after initial platelet activation (Figure 4B).
Sulfatides Enhance the Formation of Platelet–Leukocyte Conjugates in an Ex Vivo Thrombosis Model
We also examined the influence of sulfatide micelles on the formation of platelet–leukocyte aggregates in whole blood using the ex vivo thrombosis model. Sulfatides (75 μg/mL) enhanced the formation of platelet–leukocyte aggregates induced by the addition of 3 μmol/L ADP, whereas control phosphatidylcholine micelles had no enhancing effect on platelet–leukocyte aggregation (Figure 5A and 5B). The formation of platelet–leukocyte aggregates induced by ADP increased progressively over time, as also shown by Konstantopoulos et al for shear-induced platelet–leukocyte aggregation.17 At 30 minutes after initial activation with ADP, sulfatide micelles enhanced the formation of platelet–leukocyte aggregate by 70% (Figure 5A). The addition of sulfatide micelles to whole blood not previously activated by ADP had no significant enhancing effect, suggesting that an activated state of the platelets is necessary for sulfatides to enhance the formation of platelet–leukocyte aggregates. To confirm a role of sulfatides in the formation of platelet–leukocyte aggregates, we examined the effect of sulfatide antagonist MCSP on sulfatide-induced platelet–leukocyte aggregation. MCSP inhibited the sulfatide-induced formation of platelet–leukocyte aggregates by 62% at 15 minutes after the addition of ADP (Figure 5C).
Figure 5. Effect of sulfatides on platelet–leukocyte aggregation in whole blood using an ex vivo thrombosis model. A, Conditions were similar to those described in Figure 4, and platelet–leukocyte aggregation in presence of either phosphatidylcholine or sulfatide micelles was measured after addition of 3 μmol/L ADP using flow cytometry. Sulfatide micelles (?), phosphatidylcholine micelles (), PBS (), sulfatide micelles without ADP (), and phosphatidylcholine micelles without ADP (). B, Representative immunofluorescence histogram shows leukocytes that expressed platelet marker anti-CD41a–PE fluorescence above a background level (101 fluorescence) at 30 minutes after addition of buffer (white), ADP with phosphatidylcholine (75 μg/mL) (gray), or ADP with sulfatides (75 μg/mL) (black). C, Platelet–leukocyte aggregation in citrated whole blood after addition of sulfatides (50 μg/mL) and 3 μmol/L ADP in the presence of either control (annexin V, 20 μg/mL) or MCSP (20 μg/mL) in the aggregometer is shown. Aliquots of whole blood were taken at different time intervals, and platelet–leukocyte aggregation was measured by flow cytometry. Control annexin V (?), MCSP ().
Discussion
In this study, we show that the interaction between sulfatides and P-selectin leads to further platelet activation, as determined by the increase in surface expression of P-selectin and activated GPIIb/IIIa (PAC-1 epitope) on platelets, and the formation of platelet and platelet–leukocyte aggregates. Monoclonal anti–P-selectin antibodies inhibited these effects, suggesting a signal-transducing role for P-selectin in platelets similar to the one recently described for L-selectin in neutrophils.18 The platelet activating effect of sulfatides provides a positive feedback loop that potentiates formation of stable platelet aggregates and platelet–leukocyte aggregates. This amplification mechanism may be important for the formation of stable platelet thrombi at sites of vascular injuries, especially in the arterial circulation. The addition of sulfatides to unstimulated blood had no significant effect, suggesting previous activation of platelets (with P-selectin expression) is necessary for sulfatides to exhibit their activating effects. A similar activation-dependent proaggregatory effect of sulfatides was seen in platelet and platelet–leukocyte aggregation under more physiological conditions of flowing blood in an ex vivo thrombosis model. Unlike the P-selectin expression in platelets, the expression of the leukocyte ligand for sulfatides, L-selectin, is independent of its activation state. Our finding that previous activation of platelets by ADP is necessary for the proaggregatory effects of sulfatides on platelet and platelet–leukocyte aggregation suggest that sulfatides mediate their effect via platelets and not leukocytes. These proaggregatory effects of sulfatides were inhibited by the sulfatide antagonist MCSP.
The newly described role of sulfatides in platelet–leukocyte aggregation is of importance, because platelet-leukocyte aggregates are recognized to have proinflammatory properties, and increased levels are associated with an increased risk for the formation of arterial thrombi. Recent studies showed increased levels of platelet–leukocyte aggregates in patients with unstable angina,19 and after coronary angioplasty.20 Platelet–leukocyte aggregates were also noted to be an early indicator of acute myocardial infarction.20
Recently, we have shown that platelets express sulfatides on their surface, which increase after activation.5 Another study has shown that granulocytes express cell surface sulfatides and excrete sulfatides into medium.4 Thus, both excreted or surface-expressed sulfatides on platelets and granulocytes may enhance platelet and platelet–leukocyte aggregation via P-selectin. Consistent with this, it has been shown that anti–P-selectin antibodies inhibited both platelet–platelet5,12 and platelet–leukocyte aggregation.21
The sulfatide–P-selectin interactions may also be important in the adhesion of platelets to sulfatides on certain cancer cells5 or in atherosclerotic lesions,22 both of which are associated with prothrombotic conditions. The cell surface sulfatides may provide a nidus for adhesion and activation of both platelets and leukocytes, thereby contributing to the pathophysiological mechanisms of the coronary syndrome.
Sulfatide and sialyl lewis x analogs have been used in vivo to block selectin functions. In animal models, these compounds inhibit P-selectin–dependent organ injuries.23 Inhibition of P-selectin function also had antithrombotic effects in many studies. P-selectin inhibition not only accelerated thrombolysis in a primate model of arterial thrombosis24 but also reduced the extent of venous thrombosis in another primate model25 and in recurrent coronary arterial thrombosis in dogs.26 The observed beneficial effects of P-selectin blockade may be caused by the inhibition of sulfatide–P-selectin interactions in platelet and platelet–leukocyte aggregation. However, so far no clinical studies have been reported examining the effect of P-selectin inhibition on hemostasis and thrombosis.
In summary, we show that sulfatides activate platelets via P-selectin and enhance platelet and platelet–leukocyte aggregation. This mechanism may play a significant role in arterial thrombosis, in which platelet and platelet–leukocyte thrombi are formed. Consequently, therapeutic interventions directed against sulfatides or P-selectin may be beneficial in the treatment of arterial thrombosis.
Received April 26, 2004; accepted October 15, 2004.
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