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【摘要】
Objective- The objective of this study is to delineate the molecular mechanisms responsible for Staphylococcus aureus -platelet adhesion as a function of physiologically relevant wall shear stresses.
Methods and Results- A parallel plate flow chamber was used to quantify adhesion of wild-type, Spa -, ClfA - and SdrCDE - strains to immobilized platelet layers. In the absence of plasma, adhesion increases with increasing wall shear rate from 100 to 5000 seconds -1. The presence of plasma significantly enhances adhesion at all shear levels. Addition of exogenous fibrinogen yields adhesion levels similar to plasma in the lower shear regimes, but has a diminishing effect on potentiating adhesion at higher shear rates. Alternatively, as shear rate increases von Willebrand factor (VWF) plays an increasingly significant role in mediating binding.
Conclusions- Addition of plasma proteins potentiates S aureus -platelet interactions at all shear rates examined. Whereas fibrinogen plays a significant role in all shear regimes, VWF mediation becomes increasingly important as wall shear rate increases. Fibrinogen binding is dependent on bacterial adhesins ClfA and SdrCDE whereas Spa is the dominant receptor for VWF.
Addition of plasma proteins potentiates S. aureus -platelet interactions at all shear rates examined. While fibrinogen plays a significant role in all shear regimes, VWF mediation becomes increasingly important as wall shear rate increases. Fibrinogen binding is dependent on bacterial adhesins ClfA and SdrCDE whereas Spa is the dominant receptor for VWF.
【关键词】 Staphylococcus aureus protein A clumping factor A serineaspartate repeats von Willebrand factor fibrinogen
Introduction
S taphylococcus aureus is found naturally on the skin and in the nasopharynx of the human body and is known to cause blood-borne infections such as acute infective endocarditis and septicemia. 1,2 Such infections are a growing concern because of the increasing antibiotic resistance of S aureus. 1,3 Specifically, vancomycin resistance (the antibiotic of "last resort") was first reported in 1996. 4 A recent fact sheet (2003) by the Centre for Disease Control (CDC) has reported a total of 10 separate cases in the United States, thereby suggesting increasing vancomycin resistance. 5 Hence, alternative approaches must be developed to prevent and treat staphylococcal infections in the future.
The initial step in pathogenesis of infections is often cell adhesion, which is mediated by surface adhesins called MSCRAMMs ( M icrobial S urface C omponents R ecognizing A dhesive M atrix M olecules). 6,7 Adhesion is then followed by bacterial invasion and colonization of the host tissue. 6 In vascular infections the presence of blood components may influence these processes. For example, the initiation of infective endocarditis has been described as a complex stepwise process in which initial endocardial damage is followed by the deposition of platelets. 8,9 Blood-borne pathogens can adhere to this traumatized site via surface adhesins, potentially developing into mature vegetations. 8 Therefore, in order to combat such infections it is critical to evaluate which staphylococcal adhesins will support stable binding to the extracellular matrix and platelets under conditions representative of the vasculature.
S aureus is known to express numerous adhesins. 7,10 In particular, ClfA is known to participate in the infection process by facilitating bacterial binding via soluble or immobilized fibrinogen. 2 Because fibrinogen plays a significant role in platelet thrombus formation, it is likely that ClfA may be involved in supporting bacterial-platelet interactions. 11,12 This hypothesis is supported by in vivo data that show ClfA as a virulence factor in S aureus -promoted rat endocarditis. 13 In addition to ClfA, staphylococcal protein A, or Spa, is well known for the binding ability of its immunoglobulin Fc region. Spa has also demonstrated affinity with soluble and immobilized von Willebrand factor (VWF) and has been identified as a novel staphylococcal adhesin. 14 In addition to both ClfA and Spa, 3 genes encoding the serine-aspartate (SD) repeat containing proteins SdrC, SdrD and SdrE have been found in S aureus. 15 The function of these Sdr proteins is not known, but it has been hypothesized that they mediate interactions of S aureus with the extracellular matrix. 16
In the vasculature, cells are constantly exposed to hemodynamic shear forces as a result of blood flow. In the presence of shear, cell attachment to vessel walls depends on the balance between dispersive hydrodynamic forces and the adhesive forces generated by the interactions of membrane-bound receptors and their ligands. 17 In vitro studies have shown that shear stress significantly affects the molecular mechanisms of platelet adhesion and aggregation at the vessel wall. 18,19 For example, plasma protein fibrinogen mediates platelet aggregation in the low shear regime, whereas VWF mediates platelet adhesion and aggregation in high shear regimes. 14,17,20,21 It is therefore likely that the mechanisms responsible for S aureus -platelet binding will also vary depending on the shear environment. However, previous studies investigating S aureus -platelet binding mechanisms have been performed under static conditions and do not account for a potential shear force effect. 1,22
In order to closely mimic the in vivo environment in vitro, we use collagen type I to support deposition of a platelet layer. This methodology allows for the study of S aureus adhesion mechanisms to immobilized platelets under shear stress conditions relevant to the vasculature. In this study, we compare the role of exogenously added fibrinogen and VWF versus blood plasma in mediating S aureus -platelet adhesion as a function of the shear environment and identify the S aureus adhesins responsible for fibrinogen binding. This study is the first to simultaneously investigate the role of adhesins SdrCDE and ClfA in fibrinogen bridged interactions and Spa in VWF bridged interactions with immobilized platelets under physiologically relevant wall shear rates. Elucidation of such binding mechanisms governing S aureus -platelet interactions as a function of vascular wall shear rates may ultimately lead to the design of novel strategies to treat blood-borne staphylococcal infections.
Methods
Reagents and Blocking Molecules
Dulbecco?s phosphate buffered saline (DPBS) was purchased from Invitrogen Corporation. Tryptic soy broth was purchased from Difco. Fluorescein 5-isothiocyante (fluorescein isothiocyanate) was purchased from Molecular probes. Soluble VWF (VWF: Factor VIII free) was purchased from Hematologic Technologies Inc. The nonpeptide small molecule platelet IIb ß 3 antagonist XV454 23 was a kind gift by Dr Shaker A. Mousa (Albany College of Pharmacy and Pharmaceutical Research Institute; Albany, New York). The purified mouse IgG monoclonal antibodies HIP1 anti-CD42b (GPIb) and mouse IgG 1 isotype were purchased from BD biosciences (San Diego, Calif). All other reagents were purchased from Sigma (St. Louis, Mo).
Bacterial Strains, Growth Conditions and Staining Procedures
S aureus Newman WT and its mutant strains DU5873, DU5852, DU5973, DU5976 and DU5995 were provided by Dr Timothy Foster (Trinity college, Dublin, Ireland). Newman WT expresses several MSCRAMMs including protein A (Spa), clumping factor A (ClfA) and serine-aspartate repeats (SdrCDE) and can bind a multitude of plasma proteins. 7,9,16,24 DU5873 lacks Spa expression, DU5852 lacks ClfA expression, DU5973 lacks SdrC, SdrD, and SdrE genes and DU5995 lacks both ClfA and SdrCDE (summarized in the Table ).
S aureus Bacterial Strains Studied and Their Receptor Expressions
S aureus cultures were started by inoculation from glycerol stocks (-80°C) into tryptic soy broth and maintained at 37°C under constant rotation. Cells were harvested during late growth phase at 17 to 18 hours of growth (OD 600 6, cell number 3*10 9 ) and resuspended in DPBS +0.1% NaN 3. Fluorescein isothiocyanate-labeled cells were adjusted to a concentration of 10 7 cells/mL (dilution factor 300) in DPBS buffer +0.2% BSA.
Preparation of Platelet Coated Coverslips for Adhesion Studies
Washed platelets were prepared as previously described from platelet-rich plasma. 25 Platelet-poor plasma (PPP) was obtained by centrifugation of the blood at 1900 g for 15 minutes. Acid soluble collagen type I from calf skin (1 mg/mL) was first coated on the glass coverslip for 90 minutes, 1,22 followed by addition of BSA (0.1% for 30 minutes) and then incubated with washed platelets (30 minutes) to yield high platelet deposition (50 050±5000 platelets/mm 2, n=3). To assess nonspecific adhesion between perfusing bacteria and the substrate, a cell suspension of Newman WT was perfused over glass +0.1% BSA, and glass+collagen +0.1% BSA at 300 and 2000 seconds -1 (data not shown). Newman WT lacks the collagen binding adhesin and hence cannot adhere to collagen. Negligible levels of bacterial adhesion were monitored on BSA/collagen and BSA covered glass slides (data not shown), indicating BSA is an effective blocker of nonspecific adhesion. 1,22 The previously prepared immobilized platelet layer was then treated with either a physiological concentration of fibrinogen ( 9 µmol/L), VWF (7.5 µg/mL) or PPP for 30 minutes. For inhibition studies using XV454 23 or the GPIb antibody, immobilized platelets were first incubated with PPP for 30 minutes. The platelet layer was then incubated with either XV454 (1 µmol/L for 10 minutes) or GPIb antibody (40 µg/mL for 10 minutes) or a mixture of both XV454 (1 µmol/L)+GpIb antibody (40 µg/mL) for 10 minutes. For the Arg-Gly-Asp-Ser (RGDS) blocking studies, 26 immobilized platelets were first treated with RGDS peptide (150 µmol/L for 10 minutes) followed by incubation with fibrinogen. All slides were incubated in a humid environment and the excess protein washed off before the next step.
Real-time adhesion studies were performed as previously described using a parallel plate flow chamber and fluorescence video microscopy. 1,22 Firmly adherent cells were considered as those that remained stationary for at least 10 seconds at experimental end points. All experiments were performed with a minimum of 3 blood donors. Platelet coverage was not affected by flow during the experiments. 25
Calculation of % Adhesion
The % adhesion for all blocking experiments was calculated using the following equation :
The control run refers to experiments performed using either plasma or fibrinogen incubated platelet layers in the absence of blocking molecules.
Statistical Analysis
Statistical significance was performed using the single-factor analysis of variance (ANOVA) technique. In all cases, a probability value of either 0.05 corresponding to 95% or 0.01 corresponding to 99% confidence was used to test for significance. All data points represent mean values for at least 3 experimental replicates±SEM.
Informed consent was obtained from each human blood donor and the University of Maryland Baltimore County Institutional Review Board for human subject?s guidelines was followed.
Results
Hydrodynamic Shear Influences S aureus Adhesion to Immobilized Platelets
To study the adhesion of S aureus to platelets in a plasma-free shear environment, we perfused bacteria suspended in DPBS over an immobilized layer of washed platelets. All strains used lack the collagen binding receptor, thereby ensuring that measured binding events are not attributable to S aureus -collagen interactions. Figure 1 demonstrates that S aureus Newman WT adheres to immobilized platelets at all wall shear rates investigated, thereby suggesting the existence of a direct binding mechanism or a mechanism bridged by endogenous platelet proteins. The effective adhesion rates for Newman WT are significantly higher than the ClfA - mutant at all wall shear rates examined ( Figure 1 a), suggesting a critical role for ClfA at all shears studied. Interestingly, the effective adhesion rates for the ClfA - mutant decreased with increasing wall shear rate, suggesting a ClfA contribution at the higher shear rates. In contrast, the effective adhesion rates for the Spa - mutant are similar to Newman WT strain, except at the very high shear rate of 5000 seconds -1, suggesting a role for Spa primarily in the high shear regime ( Figure 1 b). The rate of detachment was found to be negligible over the range of wall shear rates investigated in all cases (data not shown).
Figure 1. Effective adhesion rate of (a) Newman WT (black bar) versus DU5852 (gray bar) and (b) Newman WT (gray bar) versus DU5873 (open bar) to washed platelets at physiological wall shear rates. Data represent mean±SE of 3 to 20 experiments for 3 or more blood donors at 37°C. * denotes P <0.05 and ** denotes P <0.01 versus the wild-type control.
Exogenous Fibrinogen Bridges S aureus Adhesion to Immobilized Platelets Under Hydrodynamic Shear
Previous studies have demonstrated S aureus -binding to molecules from both plasma and the extracellular matrix (eg, fibrinogen, VWF, collagen, fibronectin, elastin or vitronectin). 12,14 Hence, we compared S aureus adhesion to immobilized washed platelets incubated with plasma or purified fibrinogen as a function of physiologically relevant wall shear rates. This enabled us to investigate the role of exogenous fibrinogen as a bridging protein and to determine whether other plasma proteins may also serve this role. At the wall shear rates of 300 and 1000 seconds -1 S aureus adhesion to platelets incubated with either plasma or fibrinogen increased in comparison to monolayers without protein addition ( Figure 2 ). At the wall shear of 300 seconds -1 similar levels of adhesion were observed for plasma or purified fibrinogen addition suggesting that fibrinogen is the primary protein responsible for bridging in lower shear regimes. However, at 1500 seconds -1 a statistically significant increase in S aureus adhesion is observed to platelets incubated with plasma in comparison to those incubated with fibrinogen, suggesting the potential involvement of other adhesive proteins in S aureus -platelet binding at arterial shear rates.
Figure 2. Effective adhesion rate of Newman WT to washed platelets (black bar) and platelets incubated with exogenous fibrinogen (gray bar) or plasma (open bar) at physiological wall shear rates. Data represent mean±SE of 8 to 20 experiments for 3 or more blood donors at 37°C. ** denotes P <0.01 versus control (No exogenous fibrinogen or plasma addition).
S aureus Adhesins ClfA and SdrCDE are Involved in Fibrinogen Binding to Platelets at Lower Shear Regimes
To investigate the molecular mechanisms of fibrinogen bridging and the specific role of MSCRAMM ClfA, we used Newman WT and DU5852 to quantify S aureus adhesion to immobilized platelets at the wall shear rates of 300, 1000 and 1500 seconds -1. As shown in Figure 3, addition of exogenous fibrinogen significantly increased the adhesion of Newman WT at 300 and 1000 seconds -1, whereas the effect was not significant at 1500 seconds -1. Surprisingly, exogenously added fibrinogen also enhanced the adhesion of the ClfA - mutant at low (300 seconds -1 ) shear rate but failed to increase adhesion at the intermediate (1000 seconds -1 ) and high (1500 seconds -1 ) shear rates. This result suggests the involvement of a fibrinogen-binding adhesin other than ClfA that is capable of mediating adhesion events at the lower shear levels. In order to determine what other S aureus adhesin may be responsible for fibrinogen binding, the SdrCDE negative mutant, DU5973, was investigated. Fibrinogen enhanced the adhesion of SdrCDE - mutant at the low ( P =0.071 for absence versus presence of exogenous fibrinogen) and intermediate ( P =0.037 for absence versus presence of exogenous fibrinogen) shear rates but failed to enhance adhesion at the high wall shear rate. Exogenous fibrinogen failed to enhance adhesion of the double mutant ClfA - /SdrCDE - at all investigated wall shear rates. The results indicate that fibrinogen serves as a bridging molecule between S aureus and platelets at the lower wall shear rates, and that both ClfA and SdrCDE are important adhesive proteins on the bacterial surface in this shear range. DU5995, the double mutant of ClfA - /SdrCDE -, shows nearly zero binding at 1500 seconds -1 both in the presence and absence of fibrinogen even after increasing the bacterial cell suspension perfusion times to 10 minutes. Although the addition of purified fibrinogen does not increase adhesion to platelet monolayers at 1500 seconds -1, both ClfA and SdrCDE are necessary for stable binding to occur.
Figure 3. Effective adhesion rate of various S aureus Newman strains to washed platelets in the absence or presence of exogenous fibrinogen. Adhesion of Newman WT (black bar), WT+fibrinogen (stippled bar), DU5852 (horizontally striped bar), DU5852+fibrinogen (open bar), DU5973 (diagonally striped bar), DU5973+fibrinogen (checkered bar), DU5995 (gray bar) and DU5995+fibrinogen (vertically striped bar) to immobilized platelets at various wall shear rates. Data represent mean±SE of 3 to 20 experiments for 3 or more blood donors. * denotes P <0.05 and ** denotes P <0.01 versus control (No Fg incubation). denotes P <0.05 and denotes P <0.01 versus control (similarly treated Newman WT strain).
Role of VWF in Mediating S aureus Adhesion to Immobilized Platelets
Exogenously added purified VWF significantly increases adhesion of Newman WT at the wall shear rates of 300 and 2000 seconds -1 ( Figure 4 ). This increase is more pronounced at the higher wall shear rate of 2000 seconds -1. VWF failed to significantly promote adhesion of the Spa - mutant strain at either shear rate. Altogether, the results indicate an increasingly important role for both VWF and Spa in S aureus -platelet adhesive interactions as shear levels increase.
Figure 4. Effective adhesion rate of S aureus Newman and DU5873 to washed platelets in the absence or presence of exogenous VWF. Adhesion of Newman WT (black bar), WT+VWF (gray bar), DU5873 (open bar) and DU5873+VWF (stippled bar) to immobilized platelets at wall shear rates of 300 and 2000 seconds -1. Data represent mean±SE of 3 to 8 experiments for 3 or more blood donors. * denotes P <0.05 versus control (No VWF incubation).
Platelet Blocking Strategies Inhibit S aureus Adhesion to Immobilized Platelets
To verify the role of platelet IIb ß 3, a receptor complex known to bind fibrinogen and VWF in platelet aggregation, immobilized platelets were pretreated with nonpeptide antagonist XV454 ( Figure 5 a) or peptide RGDS ( Figure 5 b). 23 Both XV454 and RGDS inhibited adhesion to immobilized platelets at all shears investigated, with a maximum inhibition of 60% observed at 1500 seconds -1. Increasing the XV454 or RGDS concentration failed to further increase the inhibitory effect (data not shown). Results from control experiments using irrelevant IgG antibodies showed no inhibitory effect.
Figure 5. Effect of platelet blocking strategies on the adhesion of Newman WT to immobilized platelets. A, % adhesion of Newman WT to washed platelets incubated with exogenous plasma (black bar), plasma +20 µg/mL irrelevant antibody (gray bar), plasma +1 µmol/L XV454 (open bar), plasma +40 µg/mL anti-GPIb (checkered bar) or plasma +1 µmol/L XV454+40 µg/mL anti-GPIb (horizontally striped bar) or B, % adhesion of Newman WT to immobilized platelets incubated with exogenous fibrinogen (black bar), fibrinogen +20 µg/mL irrelevant IgG antibody (gray bar) or fibrinogen +150 µmol/L RGDS (open bar). Data represent mean±SE of 3 to 8 experiments for 3 or more blood donors at various wall shear rates at 37°C. ** denotes P <0.01 versus controls (absence of blocking molecules).
Because our data indicate the role of VWF is most significant under high shear conditions, the contribution of platelet GPIb was investigated at 1500 seconds -1 using an antibody known to block the GPIb-VWF interaction. However, the use of anti-GPIb alone did not result in significant binding inhibition ( Figure 5 a). In addition, using a mixture of XV454+anti-GPIb failed to yield further inhibition compared with XV454 alone. To verify this result at extremely high shear conditions, blocking experiments were also performed at 5000 seconds -1 with similar results (data not shown).
Discussion
The rates of nosocomial (hospital-) and community-acquired Saureus bacteremia have increased significantly since 1980. 27 Saureus is now the leading pathogen causing endovascular bacterial infection and infectious endocarditis. 28 Antibiotic resistance is compounding the growing problem, emphasizing the need to investigate molecular mechanisms governing S aureus pathogenesis. Pathogenesis begins with initial endocardial damage, exposing the underlying extracellular matrix proteins such as collagen type I, followed by deposition of platelets onto the damaged endocardium. 23 S aureus, if present in the bloodstream, may then adhere to the deposited platelets directly or via interactions bridged by plasma proteins. These events in conjunction with bacterial proliferation may eventually lead to the formation of macroscopic endocardial vegetations and metastatic seeding to other sites through the bloodstream. 1,8 The experimental model used in this study mimics the initial steps in the formation of such an infection.
Pathogenesis depends on complex interactions between Saureus, platelets and plasma proteins, which are subjected to vascular wall shear rates. 29,30 The wall shear rates evaluated in this report are representative of those found in the vasculature: 40 to 2000 seconds -1. 1,31 The increasing adhesion observed for the WT strain with increasing wall shear rate ( Figure 1 ) is likely attributable to an increase in S aureus cell flux to the immobilized platelet surface. Such a trend has previously been observed for S aureus adhesion on collagen 22 and fibrinogen 32 coated surfaces. However, in this study a decrease in adhesion rate is not observed within the range of physiological wall shear rates examined. This result suggests the possibility that multiple adhesion mechanisms may be acting to support bacterial-platelet binding.
On addition of purified fibrinogen to the immobilized platelet layer, maximum adhesion is observed at a wall shear rate of 300 seconds -1 ( Figure 2 ), which is representative of the lower range of arterial wall shear rates. This result is consistent with previous studies that demonstrated maximal adhesion efficiency (6% to 10%) of activated platelets to fibrinogen-beads at shear rates of 100 to 300 seconds -1 and decreasing efficiency up to 2000 seconds -1. 33 Platelet aggregation studies have also shown a primary role for fibrinogen mediation in the low shear regime. 21,34 Furthermore, the similar adhesion levels observed for incubation with purified fibrinogen or plasma at 300 seconds -1 suggest that fibrinogen plays the dominant role in potentiating Saureus -platelet binding at low shear 300 seconds -1, other plasma proteins in addition to fibrinogen appear to play a role in mediating S aureus adhesion to platelets. Specifically, our data suggest that VWF in particular is involved in mediating S aureus -platelet interactions under high shear conditions representative of the upper range of arterial wall shear rates ( Figure 4 ). Plasma protein VWF extends from a compacted shape under static conditions to a more outspread form with increasing shear stresses, increasing the available binding sites for S aureus. 35 The importance of VWF in mediating platelet adhesion and thrombus formation at high wall shear rates has also been previously documented and is consistent with the data presented here. 12,36
Stable cell adhesion requires membrane bound bacterial adhesins to bind ligands in the host. Newman WT adheres to immobilized platelets in the absence of any exogenous protein at all wall shear rates investigated ( Figure 1 ). Possible adhesion mechanisms may be (1) a direct receptor-ligand interaction such as between ClfA and platelet receptor IIb ß 3 and (2) an interaction bridged by proteins such as Fg or VWF secreted from platelet -granules. 8,11 Of these 2 mechanisms, the bridged interaction is more likely based on the enhanced adhesion observed in the presence of exogenous proteins. The significantly reduced adhesion for the ClfA - mutant strain highlights the importance of the ClfA receptor, which has been shown to play a probable role in wound and foreign body infections. 2,9 Although deficient in ClfA, the mutant strain expresses other surface-associated receptors such as Spa, which can still interact with immobilized platelets. The interactions may either be direct (Spa-gC1qR/p33) or via secreted -granule proteins such as VWF (Spa-VWF-GPIb/Ix or Spa-VWF- IIb ß 3 ). 12 The significant reduction in adhesion observed at 5000 seconds -1 for the Spa - mutant suggests an important role for this bacterial receptor primarily under high shear conditions ( Figure 1 b). Exogenously added VWF can also serve as a bridge between S aureus receptor Spa and platelets becoming increasingly important as wall shear rate increases ( Figure 4 ). Because Spa is known to bind VWF, 14 these data are consistent with the results described above.
In addition to VWF, exogenously added fibrinogen also enhances S aureus adhesion to immobilized platelets as a function of hydrodynamic shear. As shown in Figure 3, the importance of ClfA in fibrinogen binding is clear from data obtained with the ClfA - (DU5852) mutant strain. This result is consistent with previous studies in the literature. 10,13 However, significant adhesion was still present in the absence of ClfA indicating the potential involvement of other fibrinogen-binding receptors in the adhesion process. Hence, the ClfA - /SdrCDE - (DU5995) double mutant was studied, and a further decrease in adhesion observed in comparison to the ClfA - strain. These data indicate that both ClfA and SdrCDE are involved in fibrinogen-bridged S aureus -platelet interactions. The data also suggest a possible direct binding mechanism involving SdrCDE, because a decrease in adhesion is observed with this single mutant compared with the WT strain in the absence of exogenous protein addition. However, the involvement of endogenously released fibrinogen cannot be excluded. At a shear rate of 1500 seconds -1 the double mutant of ClfA and SdrCDE showed nearly "zero binding" in both the presence and absence of exogenously added fibrinogen to the platelet layer, thereby suggesting that both ClfA and SdrCDE are required on the bacterial surface for stable adhesion to platelets in the high shear regime.
S aureus MSCRAMMs can bind platelet integrin IIb ß 3 via a multitude of soluble plasma proteins. 33 In this study, both XV454 and RGDS function as effective blocking molecules, significantly reducing S aureus adhesion to immobilized platelets confirming a significant role for IIb ß 3. Although the binding of VWF to platelet GPIb is known to be an early event in thrombus formation at high shear, 37 no significant inhibition was noted with an anti-GPIb monoclonal antibody in S aureus binding to immobilized, activated platelets under shear. We believe that the lack of an inhibitory effect may be attributed to either the binding of VWF to collagen 38 or to the shedding of GPIb from the surface of activated platelets. Interestingly, the pharmacological intervention using anti-GPIb was effective in interfering with S aureus binding to resting platelets in bulk suspension subjected to high shear. 12 Taken together these results suggest IIb ß 3 as the primary platelet receptor mediating adhesion to S aureus. Direct S aureus -platelet binding mechanisms are also known to exist 11,39 and may be responsible for the remainder of the observed binding.
Our findings support the hypothesis that distinct pathogenic mechanisms exist in different physiological shear regimes. This general paradigm is not new, and a significant body of literature suggests that this is the case for platelet thrombus formation. For example, mechanisms governing platelet adhesion to collagen and the vascular subendothelium at shear rates 800 seconds -1 are distinct from those operative at higher shear rates. 40 However, shear modulation of the binding mechanism has not been previously demonstrated for S aureus -platelet binding. Based on the findings presented here, fibrinogen significantly enhances Saureus adhesion to immobilized platelets in the low shear regime. This is consistent with previous observations suggesting a role for fibrinogen in fundamental construction of mural thrombi under low shear conditions. 34,41 At the higher shear rates VWF significantly enhances S aureus adhesion to immobilized platelets, a result consistent with the role of VWF in thrombosis and hemostasis under high shear conditions. 12,36 The role of VWF in modulating high shear interactions has been previously described using a "two-step" process. 12 Specifically, VWF may facilitate initial bridge formation between platelet IIb ß 3 and Spa, thereby increasing the cell-cell contact time, and allowing fibrinogen to form a stable bridge between platelet IIb ß 3 and ClfA. Hence, at higher wall shear rates, VWF may be the essential bridging protein required for optimal recruitment of Saureus by platelets.
Bacterial adhesion is a prerequisite event for infection formation. This study is the first to identify a role for SdrCDE in fibrinogen-mediated adhesion to platelets and confirms the important role of ClfA in these interactions. Our study, while covering the range of physiological and pathological shear rates prevalent in the vasculature, suggests a critical role for fibrinogen at all wall shear rates. However, at the higher wall shear rates plasma VWF plays a more significant role involving bacterial receptor Spa, thereby supporting the concept of a 2-step adhesion mechanism. The shear-dependent mechanism proposed here for S aureus -platelet adhesion provides a rational basis for treatment strategies aimed to alter S aureus -platelet interactions. Combined, these results suggest therapeutic strategies that simultaneously target ClfA, SdrCDE and Spa may prevent adhesion to platelets in both high and low shear regimes of the vasculature.
Acknowledgments
Sources of Funding
This study was supported by the National Institutes of Health grant R01HL066453.
Disclosures
None.
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作者单位：Department of Chemical and Biochemical Engineering (N.P.E.G., Q.W., P.K.S., J.R.), University of Maryland Baltimore County, Baltimore, Md; and the Department of Chemical and Biomolecular Engineering (K.K.), The Johns Hopkins University, Baltimore, Md.