|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 September 2006, Vol. 108, No. 6, pp. 1903-1910. Prepublished online as a Blood First Edition Paper on June 13, 2006; DOI 10.1182/blood-2006-04-011551.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Activation-independent platelet adhesion and aggregation under elevated shear stressFrom the Roon Centre for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA.
Platelet aggregation, which contributes to bleeding arrest and also to thrombovascular disorders, is thought to initiate after signaling-induced activation. We found that this paradigm does not apply under blood flow conditions comparable to those existing in stenotic coronary arteries. Platelets interacting with immobilized von Willebrand factor (VWF) aggregate independently of activation when soluble VWF is present and the shear rate exceeds 10 000 s–1 (shear stress = 400 dyn/cm2). Above this threshold, active A1 domains become exposed in soluble VWF multimers and can bind to glycoprotein Ib  , promoting additional platelet recruitment. Aggregates thus formed are unstable until the shear rate approaches 20 000 s–1 (shear stress = 800 dyn/cm.2). Above this threshold, adherent platelets at the interface of surface-immobilized and membrane-bound VWF are stretched into elongated structures and become the core of aggregates that can persist on the surface for minutes. When isolated dimeric A1 domain is present instead of native VWF multimers, activation-independent platelet aggregation occurs without requiring shear stress above a threshold level, but aggregates never become firmly attached to the surface and progressively disaggregate as shear rate exceeds 6000 s–1. Platelet and VWF modulation by hydrodynamic force is a mechanism for activation-independent aggregation that may support thrombotic arterial occlusion.
Platelets aggregate at sites of vascular injury, forming thrombi that contribute to arrest bleeding but also occlude atherosclerotic arteries causing cardiac and cerebrovascular diseases.1,2 Platelet thrombus formation is thought to occur in successive stages. First, individual platelets adhere to altered vascular surfaces and are activated, after which the integrin IIb 3 can bind plasma proteins, notably fibrinogen, von Willebrand factor (VWF), and fibronectin; these adhesive substrates immobilized on the membrane surface then recruit additional platelets, resulting in aggregation and thrombus growth.2 Such events take place in flowing blood that generates shear forces. At shear rates exceeding 1000 s–1 in the human circulation, initial platelet arrest depends on glycoprotein (GP) Ib binding to immobilized VWF even when extracellular matrices3 or vascular structures4 present multiple reactive components. Continued platelet recruitment also becomes dependent on VWF-GP Ib as growing thrombi narrow the lumen where blood flows, locally increasing the shear rate.5 Current knowledge, therefore, is that rapidly forming but short-lived VWF-GP Ib bonds can keep platelets in contact with a surface or with one another for a limited time, until additional bonds, established mostly through integrin receptors, stabilize adhesion and aggregation.3,5,6
A feature distinguishing hemostasis from arterial thrombosis is their occurrence in different hemodynamic environments. A 90% lumen reduction in a coronary artery may cause shear rates of 20 000-40 000 s–1 at or just upstream of the stenosis,7-9 values that are 100-fold higher than in the absence of obstruction10 and 10-fold higher than in microarterioles,11 where platelets participate in hemostasis after trauma. We now have identified a unique mechanism that may be relevant for initiating platelet thrombus formation under extreme hemodynamic conditions, and it involves activation-independent as well as
Blood samples and preparation of washed blood cell suspensions Blood from healthy human volunteers was drawn from an antecubital vein. For perfusion studies, blood was collected into syringes containing D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride (PPACK; final concentration, 93 µM; Bachem Bioscience, King of Prussia, PA) to prevent clotting. Prostaglandin (PG) E1 (10 µM, Sigma Chemical, St Louis, MO) and the disodium salt of ethylenediamine tetraacetic acid (EDTA, 5 mM; Sigma) were added to the blood when indicated to inhibit platelet activation and block integrin function, respectively. To prepare plasma-free blood cell suspensions, 5 parts of blood containing PPACK were mixed with 1 part of acid-citrate-dextrose (ACD; 60 mM citric acid, 85 mM sodium citrate, and 111 mM dextrose; pH 4.5). The adenosine diphosphate (ADP) scavenger apyrase (grade 7, Sigma; ATPase/ADPase ratio < 2) was added at a final concentration of 1.3 ATPase units/mL. The blood was centrifuged at 2100 g for 13 minutes at room temperature (22°C-25°C). The resultant supernatant plasma was removed from the sedimented cells, which included platelets and leukocytes on top of the erythrocyte cushion, and replaced with an equivalent volume of divalent cation-free Hepes/Tyrode buffer (17 mM Hepes [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 130 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, and 2.8 mM dextrose), pH 6.5, containing a reduced amount of apyrase (0.65 ATPase units/mL). After gentle mixing, the cell suspension was centrifuged again, and the supernatant fluid was removed and replaced by a fresh aliquot of Hepes/Tyrode buffer. This procedure was repeated twice, each time using half the amount of apyrase, and after the final centrifugation the cells were suspended in divalent cation-free Hepes/Tyrode buffer, pH 7.4, containing 50 mg/mL bovine serum albumin (BSA; Calbiochem, La Jolla, CA). The platelet count (180 000-390 000/µL) and hematocrit level (38%-43%) were adjusted to the original values in whole blood. PG E1 (10 µM) and EDTA were added when indicated. Washed platelets for labeling with different fluorochromes were obtained by centrifuging the last blood cell suspension in Hepes/Tyrode buffer, pH 6.5, at 200 g for 10 minutes. All studies involving human subjects were conducted in accordance with the Declaration of Helsinki and were approved by the Institutional Review Board of The Scripps Research Institute. Informed consent to participate in the studies was obtained from all subjects. Preparation of recombinant VWF A1 domain and purified plasma VWF multimers
A recombinant VWF fragment comprising residues 445-733 of the mature subunit,12 including the carboxyl terminal portion of domain D3, the entire domain A1, and a small amino terminal sequence of domain A2,13 was expressed and purified following a procedure described in Supplemental Document S1, available on the Blood website; see the Supplemental Materials link at the top of the online article. Plasma-derived VWF multimers were purified as previously described.14 The antibody NMC-4 (a generous gift of Dr Akira Yoshioka, Nara Medical College, Nara, Japan) was purified as reported elsewhere.15 NMC-4 binds to the VWF A1 domain and blocks binding to GP Ib Ex vivo perfusion experiments Perfusion experiments were conducted at 37°C by exposing blood to different thrombogenic surfaces, which included fibrillar type 1 collagen from bovine tendon (acid insoluble; Sigma),3 immobilized multimeric VWF6 purified from plasma as previously described,14 extracellular matrix deposited by mouse dermal fibroblasts (Document S1), and platelet thrombi formed by perfusing whole human blood over fibrillar type 1 collagen. The glass coverslips onto which thrombogenic substrates were immobilized were assembled into a parallel plate rectangular flow chamber.3 Blood was perfused through the chamber by aspiration with a syringe pump (Harvard Apparatus, Holliston, MA) at the desired flow rate. The perfusion chamber was mounted on the stage of an inverted microscope (Axiovert 135M; Carl Zeiss, Thornwood, NY) for real-time visualization of platelet interactions with the immobilized substrates and with one another. For epifluorescence microscopy, platelets were rendered fluorescent by the addition of 10 µM mepacrine (quinacrine dihydrochloride; Sigma),6 unless otherwise specified. For 2-color experiments, platelets in untreated whole blood were rendered fluorescent with mepacrine, and washed platelets (in Hepes/Tyrode buffer, pH 6.5), whose ability to be activated was blocked with 10 µM PG E1, were rendered fluorescent by incubation with calcein red-orange, AM (1 mg/mL solution in dimethyl sulfoxide; Cell Trace, Invitrogen, Carlsbad, CA), for 15 minutes in the dark. After labeling, the platelets were sedimented by centrifugation at 600 g for 8 minutes and resuspended in Hepes/Tyrode buffer, pH 7.4. For electron microscopy analysis, platelets and aggregates interacting with immobilized VWF were perfusion-fixed under flow with a buffered 4% paraformaldehyde solution. For scanning electron microscopy (JSM-6300F, JEOL, Munich, Germany) the fixed specimens were dehydrated in ethanol baths of increasing concentration (up to 100%) and then critical-point dried with CO2 and sputter-coated with platinum. For reflection interference contrast microscopy (RICM), no labeling of the blood cells was required. This technique resolves the cell membrane contact with a surface and indicates the distance between the 2 through interference colors.16,17 In our studies, interference was caused by the light reflected from the glass surface onto which the substrate was coated and that from the membranes of platelets flowing in close proximity to the coated glass or interacting with it. Since we used a black-and-white video camera, interference colors were obtained on a gray scale, in which zero-order black corresponds to a distance between platelet membrane and coated glass surface of 4 to 12 nm, and white corresponds to a distance of more than 20 to 30 nm.16,17 Structures that are separated by more than 30 nm are out of focus. Intravital microscopy in a mouse arterial injury model All animal care and experimental procedures complied with the Guide for the Care and Use of Laboratory Animals, United States Department of Health and Human Services, and were approved by the Animal Care and Use Committee of The Scripps Research Institute. The procedure to expose mesenteric vessels and visualize thrombus formation at the site of a ferric chloride–induced lesion using fluorescent platelets has been previously described18 and is reported in detail in Document S1. The microscope used was a Zeiss Axioplan2 equipped with an Achroplan 40 x/0.8 NA water-immersion objective. Image acquisition and analysis All experiments were recorded on S-VHS videotape using a VCR (SVO-9500MD; Sony, Tokyo, Japan) at the acquisition rate of 30 frames per second. Fluorescence images were acquired with a silicon-intensified (SIT) high-sensitivity camera (Hamamatsu Photonics; Bridge-water, NJ) and RICM images with a CCD (charge-coupled device) camera (DXC-390; Sony). The objectives used (all from Carl Zeiss) were Plan-Neofluar 40 x/0.75 NA (Figures 1, 2B, 3, 6), Plan-Neofluar 10 x/0.30 NA (Figure 2A), and Plan-Neofluar Ph3 Antiflex 63 x/1.25 NA oil immersion (Figure 5). Image analysis was performed offline using the Metamorph software package (Universal Imaging; West Chester, PA).6 The supporting movies available online were prepared by digitizing and editing the recorded analog tapes with Adobe Premiere (Adobe Systems, San Jose, CA).
Distinct platelet adhesion and aggregation mechanisms as a function of shear rate We first evaluated thrombus formation on a model reactive surface composed of collagen type 1 fibrils in a chamber perfused with blood containing 93 µM PPACK as the anticoagulant. At the wall shear rate of 3000 s–1, as expected, single platelets adhered and accumulated into thrombi that grew at sites of initial arrest (Figure 1; Video S1). At 24 000 s–1, in contrast, platelets appeared to be linked to one another even before stable adhesion took place, but no interplatelet links were visible by epifluorescence microscopy. The elevated fluid drag could stretch these aggregates and eventually detach them from the growing thrombus (Figure 1; Video S1). Such events were evident in the initial 20 to 40 seconds, before rapid aggregation took place, and delineated a hitherto unrecognized mechanism of platelet cohesion operating in the initial stages of thrombus formation at pathologically elevated shear rates. A peculiar feature of this form of aggregation was that linked platelets were not closely juxtaposed (Figure 1; Video S1). Activation-independent platelet adhesion and aggregation require immobilized as well as soluble VWF
Because platelet adhesion to collagen depends on bound VWF above a threshold shear rate,3 we evaluated whether immobilized VWF alone could initiate platelet aggregation in rapidly flowing blood. Moreover, since blocking platelet activation has no effect on GP Ib
As expected,6 individual rolling platelets covered the VWF surface at the shear rate of 3000 s–1. In contrast, at 20 000 s–1 rolling platelets formed aggregates within 100 µMto200 µM from the boundary where immobilized VWF was exposed to blood, equivalent to a few seconds from the initial surface contact (Figure 2A; Video S2, part 1). These aggregates grew larger while translocating in the direction of flow and had varying shape and velocity during motion, but were mostly elongated with a stringlike morphology during periods of prolonged arrest that lasted several seconds (Video S2, part 1). We then perfused a plasma-free blood cell suspension to evaluate whether addition of soluble VWF was sufficient to mediate activation-independent platelet aggregation. In agreement with the results in whole blood, at the shear rate of 3000 s–1 the VWF surface was covered by single rolling platelets in equal number whether soluble VWF was present or not (Figure 2B; Video S2, part 2). At the shear rate of 24 000 s–1, in contrast, single platelet adhesion was markedly lower than at 3000 s–1 in the absence of soluble VWF, but activation-independent platelet aggregates formed as in whole blood when VWF was added (Figure 2B; Video S2, part 2). Under extreme shear stress conditions, therefore, enhanced platelet adhesion and aggregation occurred at the interface between immobilized and soluble VWF without requirement for other plasma components. This VWF-mediated platelet aggregation was inhibited by a monoclonal antibody against the VWF A1 domain (Figure 2C) or one against GP Ib (not shown). Activation-independent platelet aggregates also formed in pulsatile flow characterized by shear rate cycles between 30 and 62 000 s–1 with a 1-second period, but were smaller when the cycle was between 30 and 57 000 s–1 and absent when the cycle was between 30 and 37 000 s–1 (Video S3). Modulation of activation-independent platelet adhesion and aggregation by shear stress
Because activation-independent platelet aggregation occurred above a threshold shear rate, we evaluated formation and stability of rolling aggregates moving across a perfusion chamber with flow velocity (shear rate) varying bidirectionally as a function of distance from the inlet and position relative to the centerline of the flow path21 (Figure 3; Video S4, part 1). We assumed that blood is a Newtonian fluid at the shear rates relevant for these studies, to which this relationship applies:
bonds are enhanced by increasing shear stress, and this positive modulation may become less reversible as the bonds are exposed to higher stress and, possibly, more interactions are established at the interface of soluble and immobilized VWF (stretched, chainlike aggregates).
Properties of interplatelet connections formed under high shear stress
The links between platelets aggregated through VWF-GP Ib
The ability of these interconnecting structures to fuse with other platelets and their elasticity suggested that they could comprise cell membrane. Evaluation of perfusion-fixed activation-independent aggregates by electron microscopy (Figure 4) demonstrated the presence of membrane protrusions connecting platelets to one another and to the surface. The connecting membrane links were longer in what appeared to be elongated (stringlike) surface-adherent aggregates (Figure 4A,B) than in ellipsoid rolling aggregates (Figure 4C). The effects of perfusion-fixation were monitored by microscopic observation; thus, we had positive evidence that the morphology of rolling aggregates was preserved after fixation. All aggregates seen during perfusion that were not in a stringlike configuration were rolling on the surface. To elucidate in real time the nature and dynamic properties of the links that support activation-independent platelet aggregation, we used RICM, a technique that visualizes areas of a cell membrane in close contact with a surface,16,17 and followed the process from the arrest of single platelets onto immobilized VWF. From a discrete point of tight adhesion, over a period of several seconds, the platelet body was stretched by the fluid drag into a structure that exceeded 10 µM in length but was less than 1 µM thick at the narrowest point (Figure 5A; Video S6, part 1). During stretching, most of the platelet body slid forward, and only a limited upstream area of the membrane provided firm attachment to immobilized VWF. Additional platelets then adhered to the ones that had become elongated on the surface. Some that were stretched by the fluid drag contributed to the increasing length of a stringlike formation, while many retained their discoid morphology (Figure 5B; Video S6, part 2). In the end, stretched platelets joined to one another formed continuous structures that reached lengths of 100 µM to 200 µM and remained attached to immobilized VWF for minutes, while hundreds of discoid platelets adhered to them and to one another with arrest times of variable duration until eventually detaching as rolling aggregates (Figure 5C; Video S6, part 3). In the absence of soluble VWF, in contrast, only single platelets interacted with immobilized VWF, and thin tethers occasionally appeared between points of tight adhesion and the moving cell body,22,23 but no interplatelet links developed, confirming the essential role of soluble VWF in the process (Video S6, part 4).
When activation was not inhibited, platelets within a stringlike aggregate that were firmly adherent to VWF were spread, while those at the downstream end, which often detached into rolling aggregates, were not (Figure 5D; Video S7). This observation is in agreement with the concept that VWF-mediated platelet aggregation can precede activation and stable surface adhesion.
Activation-independent platelet aggregation occurs on different substrates. To confirm the data obtained with immobilized collagen (Figure 1; Video S1) and demonstrate that activation-independent platelet aggregation mediated by soluble VWF could occur on several relevant surfaces, we perfused blood at varying wall shear rates over the extracellular matrix deposited by mouse skin fibroblast (Video S8) or platelet thrombi formed by a previous perfusion of whole blood over fibrillar type 1 collagen (Video S9). In the latter experiment, PG E1–treated, washed platelets labeled with a red fluorochrome were added into a washed erythrocyte suspension supplemented with purified VWF (15 µg/mL) and perfused over aggregated platelets, visualized with a green fluorochrome. In either case, activation-independent rolling platelet aggregates and more firmly adherent elongated aggregates formed at progressively increasing shear rates, in a manner similar to that observed on surface-immobilized purified VWF. Activation-independent rolling aggregates were visible on the surface of platelet thrombi at a shear rate of 10 000 s–1 (Video S9), thus lower than on purified VWF. This may be due to the fact that platelet activation results in the release of VWF stored in The function of multimeric VWF is regulated by shear stress To explain why activation-independent platelet aggregation mediated by soluble VWF occurs only above a threshold shear rate, we compared the adhesive properties of multimeric plasma-derived VWF and recombinant isolated VWF A1 domain. The latter was expressed as a dimer (dVWFA1) with interchain disulfide bonds in a portion of domain D3 preceding A1, thus reflecting the assembly of VWF multimers in this region.24-26 When added to a washed blood cell suspension perfused in a variable shear rate flow chamber (Figure 3), dVWFA1 mediated activation-independent platelet aggregation at the lower shear rates tested, well below the threshold required for function of multimeric VWF (Figure 6; Video S10). In contrast, above this shear rate threshold, multimeric VWF mediated the formation of aggregates that could become elongated and firmly adherent under the effect of increasing hemodynamic force, while dVWFA1 could no longer support platelet aggregation (Figure 6; Video S10). The same results were observed when the concentration of dVWFA1 in solution was increased by 10-fold (50 µg/mL as opposed to 5 µg/mL; Video S10), a clear indication that the functional difference between multimeric VWF and isolated A1 domain fragment was the consequence of distinct molecular architecture rather than concentration of active sites.
These findings demonstrate that A1 domain binding to GP Ib Activation-independent platelet aggregates form in vivo To evaluate whether VWF-mediated platelet aggregation occurs within the vasculature of a living animal, we performed experiments in the mesenteric circulation of anesthetized mice. PG E1–treated platelets labeled with a red fluorochrome and noninhibited platelets labeled with a green fluorochrome were injected concurrently and their accumulation monitored at a site of injury. Noninhibited platelets formed stable and compact thrombi, while PG E1–treated platelets formed loose aggregates that became stretched by flow forces while rolling over the stable thrombi (Video S11). The latter resembled the VWF-mediated aggregates observed during in vitro perfusion studies. Similar events were observed both in injured arterioles and venules with initial wall shear rates in the order of 1300 and 700 s–1, respectively, thus below the threshold that we found was required in ex vivo perfusion experiments for the induction of VWF-mediated aggregates independently of activation. These aggregates, nonetheless, appeared on the surface of thrombi, thus at sites of vascular lumen restriction where shear stress was higher than at the wall. Moreover, the thrombi were formed by activated platelets that release VWF multimers larger than those in circulating blood, which may be more active and contribute to lowering the shear stress threshold required for the formation of activation-independent aggregates.
We have identified a mechanism of activation-independent platelet aggregation mediated by VWF binding to GP Ib that occurs exclusively at the interface of immobilized and soluble VWF, is modulated by shear stress, and may thus contribute to thrombotic arterial occlusion. As delineated here, the process is distinct from previously identified mechanisms of platelet adhesion and aggregation under flow conditions. It is known that shear stress in excess of 60 dyn/cm2 can induce VWF binding to GP Ib,29 resulting in platelet aggregation, but only if activation can occur and IIb3 is fully functional.30-32 In contrast, VWF-mediated platelet aggregation as described here is independent of activation and IIb-3 function. The previously described "beads-on-a-string," that is, platelets bound to ultralarge VWF multimers on endothelial cell membranes,33 appear somewhat similar to the elongated aggregates shown here under extremely elevated shear stress but, in fact, form through a distinct process. These "strings" are newly released and extremely long VWF molecules with enhanced affinity for GP Ib,34 to which single platelets adhere at shear rates between 250 and 5000 s–1 and in the absence of plasma,33 thus of soluble VWF, 2 conditions incompatible with activation-independent aggregation. In the aggregates shown here under high shear stress, in contrast, the interplatelet connections are formed by stretched membrane segments rendered adhesive by plasma VWF multimers functionally modulated by tensile stress, which constitutes a previously unrecognized mechanism of platelet aggregation.
Our results indicate that hydrodynamic force exerts multiple effects during activation-independent platelet aggregation. A first shear stress threshold regulates the induction of platelet-platelet contacts, leading to the formation of "rolling" aggregates, in which interplatelet cohesion predominates over surface adhesion. The sequence of events in the process appears to involve single platelet tethering to immobilized VWF, binding of soluble VWF to GP Ib A higher shear stress threshold than required for induction of VWF-mediated aggregation regulates the formation of activation-independent aggregates that remain stationary on the immobilized VWF surface for prolonged periods of time. This additional effect of shear stress appears to depend on major morphologic changes of platelets. By elongating and, thus, reducing their cross-sectional area perpendicular to the direction of flow, stretched platelets are subjected to lesser hydrodynamic drag than discoid platelets. This adaptation decreases the tensile stress on adhesive bonds at points of surface contact, prolonging their lifetime. Stretched platelets also may provide an extended membrane surface for the high-density binding of VWF multimers uncoiled by shear forces,29 possibly reinforced by VWF self-association.39,40 In fact, availability of soluble VWF is a strict requirement for stretched platelets to form and act as the elastic links that support interplatelet cohesion within activation-independent aggregates anchored to the surface. In this regard, stretched platelets are distinct from single platelets with thin tethers transiently adherent to immobilized VWF,22,23 which have adhesion times of less than a second to a few seconds as opposed to minutes (Video S6, part 4). Stretching of platelets requires a point of firm attachment to the surface, which may exist only when soluble VWF associates with immobilized VWF to increase the local density of VWF A1 domain. The concept that soluble VWF can enhance the functional activity of immobilized VWF expressed through the A1 domain is in agreement with previous findings.39 Platelet stretching and the resulting amplification of adhesive properties, therefore, are expressions of a specific synergistic function of soluble and immobilized VWF and not passive adaptations to hydrodynamic force.
In conclusion, we have outlined a mechanism mediated by soluble VWF that supports the attachment of large clusters of platelets to immobilized VWF without requiring activation and
We thank Brian Savage for helpful discussion and insights, and James R. Roberts and Richard A. McClintock for technical assistance.
Submitted December 16, 2005; accepted May 8, 2006.
Prepublished online as Blood First Edition Paper, June 13, 2006; DOI 10.1182/blood-2006-04-011551.
Supported by National Institutes of Health grants HL-31950, Hl-42846, HL70818, and HL-78728.
Z.M.R. designed research, performed research, analyzed data, and wrote the paper; J.N.O. performed research and analyzed data; R.H. analyzed data; A.B.F. produced essential reagents; and A.J.R. implemented techniques, performed research, analyzed research, and reviewed the paper.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Zaverio M. Ruggeri, The Scripps Research Institute—MEM 175, 10550 North Torrey Pines Rd, La Jolla, CA 92037; e-mail: ruggeri{at}scripps.edu.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
