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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Laboratory of Blood and Vascular Biology, The
Rockefeller University, New York, NY; and the Mount Sinai School of
Medicine, New York, NY.
The conventional description of platelet interactions with
collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then
recruit additional layers of platelets. To obtain dynamic information,
we studied gravity-driven platelet deposition in vitro on purified type
1 collagen by video phase-contrast microscopy at 22°C. With untreated
human and wild-type mouse platelets, soon after the initial adhesion of
a small number of "vanguard" platelets, "follower" platelets
attached to the spread-out vanguard platelets. Follower platelets then
adhered to and spread onto nearby collagen or over the vanguard
platelets. Thus, thrombi formed as a concerted process rather than as
sequential processes. Treatment of human platelets with monoclonal
antibody (mAb) 7E3 (anti-GPIIb/IIIa ( Platelet adhesion and aggregation play important
roles in physiologic and pathologic phenomena. Thus, platelet adhesion
and aggregation after trauma to a blood vessel are crucial to stop hemorrhage and restore hemostasis, whereas adhesion and aggregation on
a damaged atherosclerotic plaque can result in thrombosis, vaso-occlusion, and distal infarction. Depending on the surface, platelet adhesion is mediated by one or more surface receptors with
affinity for one or more tissue components, including collagen, von
Willebrand factor, fibrinogen, fibronectin, and perhaps vitronectin, laminin, and thrombospondin.1
Collagen is consistently a highly thrombogenic surface coating in model
systems of platelet adhesion when judged by the rate of platelet
deposition, the ability to support the formation of multiple layers of
platelets, the strength of the adhesion, or the ability to support
fibrin formation.2-5 Platelet-collagen interactions are
complex, with the presence of multiple platelet receptors for collagen
leading to differences in behavior as a function of collagen type;
collagen architecture (monomeric or fibrillar); availability of soluble
adhesive glycoproteins such as von Willebrand factor, fibronectin, and
fibrinogen; the presence of divalent cations and their concentrations;
and shear forces.6-22 Nonetheless, the central paradigm is
that platelet interactions with collagen occur in a sequential manner
in which platelet adhesion, platelet activation and spreading, and
platelet-platelet interactions follow one another in series.
To understand better the process of platelet adhesion to type 1 collagen, the factors that contribute to the uniquely high thrombogenicity of collagen, and the role of the platelet glycoprotein (GP) IIb/IIIa ( Human and mouse GPIIb/IIIa antagonists
Collagen
Platelet preparation Approval was obtained from the Institutional Review Board for these studies. Informed consent was provided according to the tenets of the Declaration of Helsinki. Human blood (8.5 mL) was drawn after informed consent into acid-citrate dextrose (ACD-A) (1.5 mL) and was centrifuged for 3.5 minutes at 700g at 22°C to prepare platelet-rich plasma (PRP). The PRP was mixed with 0.1-vol ACD and centrifuged at 1000g for 10 minutes at 22°C; the pellet was then resuspended in 1 mL HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-modified Tyrode buffer (138 mM NaCl, 10 mM HEPES, 12 mM NaHCO3, 2.7 mM KCl, 0.4 mM NaH2PO4, 0.1% glucose, 0.35% bovine serum albumin [BSA; Fraction V; Sigma, St Louis, MO], pH 7.4; HBMT) and was gel-filtered on a column of Sepharose-2B (Pharmacia, Piscataway, NJ) using HBMT for elution. Gel-filtered platelets (GFP) were pretreated with 0.01-vol buffer, antibody 7E3 (10 µg/mL), tirofiban (0.25 µg/mL), or control mouse immunoglobulin G (IgG) (10 µg/mL; Jackson ImmunoResearch Laboratories, West Grove, PA) for 15 minutes at 22°C. MgCl2 was added at 0.01 vol to a final concentration of 2 mM. In one experiment blood was drawn into ACD from a control and from a patient with Glanzmann thrombasthenia whose platelets contained no detectable GPIIb27 but did contain trace amounts of GPIIIa.28 PRP was prepared, washed twice, and resuspended in HBMT; adjustments were then made to 100 000 platelets/µL, MgCl2 (2 mM) was added, and the platelets were allowed to adhere to a collagen-coated microchamber for 1 hour at 22°C.Alternatively, human blood was drawn into ACD and centrifuged as above. After removing the PRP, the buffy coat was removed and placed in another tube. A volume of buffer equal to the amount of PRP removed was then added to the buffy coat and to the residual red blood cells. Each tube was mixed and recentrifuged as above, and the resultant platelet-rich buffer (PRB) was removed. The PRP and PRB were pooled, and the suspension was incubated with buffer or 7E3; MgCl 2 was then added as above. Platelets from F2 wild-type mice of mixed C57Bl/6 and 129 lineages and
Microchamber A 1.25-mm-thick microchamber was constructed from a glass slide coated with chlorinated organopolysiloxane (Sigmacote; Sigma), a silicone elastomer gasket (Sylgard 184; Dow Corning, Midland, MI), and a no. 1.5 glass coverslip coated first with Sigmacote and then with 1% polystyrene in chloroform using a spincoater. The gasket was designed to produce 12 separate channels of 3.4 µL (Figure 1). The polystyrene-coated coverslip was then coated with collagen by filling the chambers by capillary action with a collagen solution (33 µg/mL in 0.05% HAc) and incubating for 1 hour at 37°C. Any residual uncoated surfaces in the channels were blocked by first rinsing and then incubating the chambers for 1 hour at 22°C in HBMT. Channels were washed with HBMT with or without 2 mM MgCl2. GFP or PRP/PRB (100 000 platelets/µL) were added to the channels by capillary action, and channels were sealed with silicone grease. Interactions between platelets and the collagen-coated surface were monitored at 22°C by microscopy, as detailed below.
Phase-contrast microscopy and recording Phase-contrast microscopy was performed with an inverted Zeiss Axiovert microscope (Carl Zeiss, Thornwood, NY) with a computer-controlled mechanical stage (Ludl Electronic Products, Hawthorne, NY) using a × 100, 1.4 NA Zeiss Neofluar objective. Images were obtained automatically and concurrently for 2 hours from each channel at 2.4-minute intervals and were stored on a video disk recorder (TW-3038F; Panasonic, Secaucus, NJ). Each acquired image was an average of 2 video frames from the camera (Hamamatsu Newvicon, Bridgewater, NY).Quantitative assessment of the pattern of platelet deposition and statistical analysis An observer masked to the platelet treatment viewed the time-lapse recordings. Using frame-by-frame analysis, the location of each new single platelet that developed stable adhesion (defined as the frame in which Brownian motion ceased and the platelet image was unobstructed) to collagen or to an already adherent platelet was recorded using Adobe Photoshop (Adobe Systems, San Jose, CA) and Equilibrium DeBabelizer Pro (Equilibrium Technologies, San Rafael, CA) software. A circle was drawn around the platelet image, and the coordinates of the center of the circle were used as the platelet position. Each subsequent frame was analyzed to document the position of each additional platelet for the 2-hour duration. After adhesion was completed, the distribution of accumulated positions was compared to that expected from a Poisson distribution. For each surface, minimum and maximum loci on the x-axis and y-axis were defined so as to identify a central area of length 600 pixels on the x-axis and height 450 pixels on the y-axis. Each area was then divided into 300 30 × 30 pixel-square grids, and the number of grids containing 0, 1, 2, or 3 or more platelets was tallied. If the positions of the platelets on the area were random, they would have conformed to the expectations under a Poisson distribution. The parameter of the putative Poisson variable was estimated as = x/300,
where x = total number of platelets in the designated area. The expected number of grids containing y platelets
(y = 0,1,2...) was obtained from the Poisson
distribution as
300(exp )(y/y!). Observed
numbers were compared with expected numbers using a  2
goodness-of-fit test. The entire analysis was repeated using 1200 15 × 15 pixel-square grids. These calculations were implemented using SAS software for the PC and by hand calculations for the goodness-of-fit tests.
Scanning electron microscopy Platelet adhesion to collagen-coated polystyrene microtiter wells (CoStar, Cambridge, MA) was conducted essentially as described. After platelets were allowed to adhere for 1 hour at 22°C, the wells were washed twice with 100 µL HBMT/MgCl2 and then with phosphate-buffered saline (PBS; 137 mM NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, 2.7 mM KCl, pH 7.4). Platelets were fixed by adding 100 µL 1.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, and incubating the mixture overnight at 4°C. Wells were then washed with cacodylate and postfixed with 1% osmium tetroxide in cacodylate for 30 minutes. Wells were dehydrated in graded steps of ethanol and then critical point dried with liquid CO2. Platelets were sputter coated with gold-palladium to 10 nm. Samples were examined in a Hitachi S530 scanning electron microscope (Tokyo, Japan).Differential interference microscopy and immunofluorescence In some experiments, the coverslip containing adherent platelets was incubated at 22°C for 30 minutes with 1 µg/mL biotinylated murine antibody to P-selectin (G1/G1-4; Ancell, Bayport, MN) and then, after rinsing, for 30 minutes with 10 µg/mL fluorescein isothiocyanate-labeled avidin (UltraAvidin Leinco Technologies, St Louis, MO). After fixing with 4% paraformaldehyde and mounting in 50% glycerol in PBS, the coverslips were visualized with a × 100 objective by differential interference microscopy and immunofluorescence (BX60 microscope; Olympus, Melville, NY), Spot RT camera (Diagnostic Instruments, Sterling Heights, MI), or Olympus PM20 photomicroscopy system.Transmission electron microscopy The hydrophobic side of Thermanox coverslips (Ted Pella, Redding, CA) was fitted with a silicone gasket to create microtiter wells. Platelet adhesion to the collagen-coated surface was conducted as described. After platelets were incubated for 1 hour, the wells were washed twice with 100 µL HBMT/MgCl2 and then were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate, pH 7.4, for 1 hour at 22°C. Wells were then washed with cacodylate buffer and postfixed with 1% osmium tetroxide for 15 minutes. The wells were dehydrated in graded steps of ethanol and polymerized with increasing grades of Epon to 100% for 60 minutes. Sections of 0.14 µm were cut perpendicular to the plane of the coverslip with a diamond knife and a Reichert-Jung Ultra Cut Microtome (Vienna, Austria). Specimens were stained with 2% aqueous uranyl acetate + 100% ethanol (1:1) for 5 minutes and then were washed with distilled water. Thereafter, specimens were stained with Reynold lead citrate for 3 minutes and washed with distilled water. Samples were then examined in a Hitachi H7000 transmission electron microscopy (TEM) microscope.
Human platelet adhesion to collagen Videomicroscopy (see the Supplemental Videos link at the top of the online article for VHS video segment A, which plays at 3 frames per second) revealed that initially a small number of platelets, which we term vanguard platelets, adhered directly to the collagen-coated surface and rapidly began to spread and lose the distinctive mound-shaped structure typical of the granulomere. Then, though additional vanguard platelets also adhered directly to the collagen, other platelets in suspension (follower platelets) became tethered to vanguard platelets by way of filopodia. We could not unequivocally determine, however, whether the filopodia were extending from the vanguard platelets, the follower platelets, or both. Follower platelets remained tethered to the vanguard platelets by way of filopodia for variable periods of time, after which they adhered to nearby collagen directly and spread or began to spread over the vanguard platelets to which they attached. This process resulted in the development of a sheetlike underlayer(s) of platelets and the growth of platelet thrombi, as islands, on top of the sheet of platelets. Ultimately, platelets spreading from different islands contacted each other, and the platelet borders became indistinct when viewed en face. Once adjoining islands made contact, there appeared to be movement of the contents of the platelets toward each other, with subsequent continued active ruffling of the edges of the interacting platelets.The adhesion of platelets to collagen was not affected by the inclusion
of 10 µg/mL control polyclonal murine IgG. In the presence of the
antibody 7E3 or tirofiban, however, in addition to an approximately
30% decrease in total platelet deposition as a result of inhibiting
the interactions between vanguard and follower platelets, there was a
marked difference in platelet behavior (VHS video segment A). Platelets
adhered to collagen, multiple filopodia extended from these platelets
onto the nearby collagen surface, and the filopodia demonstrated
dynamic movement, but spreading was more variable and overall appeared
less extensive than in the absence of 7E3. When compared to the
islandlike deposition pattern formed by untreated platelets, 7E3- and
tirofiban-treated platelets developed more uniformly scattered
patterns. The filopodia that extended out from the adherent platelets
sometimes contacted other platelets, and some platelets partially
spread over other platelets, but there was virtually no tethering of
follower platelets, platelet thrombi, or evidence of platelets acting
as an interacting mass (VHS video segment A). Thus, the platelets did
not spread in the sheetlike manner that was observed in the absence of
7E3, and the platelet granulomeres remained more prominent than in the
absence of 7E3. Even after 1 hour, the borders of the platelets continued to remain distinct. The similarity in results with 7E3 and
tirofiban support the interpretation that the effect of 7E3 is
primarily caused by its inhibition of GPIIb/IIIa. Microchamber studies
were also conducted with the platelets of a patient with Glanzmann
thrombasthenia whose platelets lacked immunodetectable GPIIb but had
readily detectable trace amounts of GPIIIa.27,28 The
pattern of platelet adhesion was indistinguishable from that of
platelets in the presence of 7E3 (Figure
2). Although we did not directly measure
the patient's platelets for
To quantitatively assess the patterns of platelet deposition, we
compared the spatial distribution of adherent platelets in each
experiment to that predicted by a Poisson distribution (Table 1). In 3 separate experiments with
control platelet preparations, values for the number of 30-pixel
squares with 0, 1, 2, and 3 or more platelets were consistent with a
Poisson distribution (P = .90, .73, and .81, respectively). In sharp contrast, all 3 platelet samples treated with
7E3 had decreased values relative to those predicted by a Poisson
distribution for the number of squares with 0, 2, and 3 or more
platelets, and increased values for the number of squares
with one platelet. In each of the 3 experiments, the deviation
of the actual distribution from that predicted by a Poisson
distribution was significant (P < .0001; and
P < .01). Similarly, the platelet sample treated with
tirofiban produced a pattern that deviated significantly from the
Poisson distribution in the same manner (P < .0001).
Similar results were obtained using 15 × 15 pixel square grids (data
not shown).
Scanning electron microscopic images of untreated human platelets after 1 hour of adhesion demonstrated a sheetlike underlayer(s) of extensively spread platelets and adherent, but unspread, follower platelets on top of the spread platelets (Figure 3A-B). In sharp contrast, the platelets treated with 7E3 did not form such a sheetlike underlayer and, even though platelets spread over each other, the borders between platelets were more distinct (Figure 3C-D). In addition, even though some platelets demonstrated a diminution in the prominence of their granulomeres, many retained their moundlike granulomeres to a greater extent than did those forming the sheetlike layer(s) in the untreated sample. Differential interference microscopy of normal human platelets revealed
images similar to those obtained by scanning electron microscopy
(Figure 4A). Immunofluorescence detection
of the
Transmission electron microscopy of cross-sections of untreated human platelets adhering to collagen (Figure 5Ai-iii) revealed that, in many areas, the sheetlike layer observed by scanning electron microscopy was actually made up of a stack of variably spread platelets whose membranes were intimately apposed (Figure 5Ai, thick arrow). Above these layers of platelets were adherent, partially spread platelets, and on the very top were follower platelets with readily detectable granulomeres (Figure 5Ai, upper thin arrow). In contrast, in the presence of 7E3 (Figure 5Bi-iii), fewer layers of platelets were observed. Even though platelet membranes were apposed or partially overlapped in some areas, the limits of the membrane of each platelet remained distinct. Moreover, a larger number of platelets directly adherent to the collagen retained their moundlike granulomeres (Figure 5Bi, iii, arrowheads). Mouse platelet adhesion to collagen Wild-type mouse platelets, though smaller than human platelets (approximately 4-5 fL vs 8-9 fL), adhered to collagen much like human platelets, though the loss of granulomere structure was not as pronounced (see VHS video segment B). Thus, vanguard platelets adhered and began to spread, and then follower platelets attached to the vanguard platelets. Follower platelets then adhered and spread, producing islands of platelet thrombi. Bridgelike filopodial extensions eventually formed between platelet islands, and this resulted in the movement of platelet material between islands and the formation of an interactive platelet mass. As with untreated human platelets, statistical analysis demonstrated that the pattern of platelet deposition conformed to that predicted by a Poisson distribution (Table 1).
Scanning electron microscopy demonstrated that wild-type mouse
platelets (Figure 6A-B), like untreated
human platelets (Figure 3A-B), spread and formed a sheetlike mass in
which the borders of the individual platelets could not be discerned.
Studies of platelet adhesion to collagen and other adhesive
proteins at select time points under static conditions, or under the
influence of variable shear forces, have demonstrated that platelets
not only adhere more rapidly to collagen-coated surfaces than other
surfaces, they also form larger thrombi.2-4,8,14 In
addition, such assays have helped to define the roles of platelet receptors that have been reported to participate in platelet adhesion to collagen or collagen-induced intracellular signaling, including To overcome this limitation, we developed a high-resolution time-lapsed
videomicroscopy system and studied adhesion of human and mouse
platelets to collagen. Our data demonstrate that in contrast to the
traditional description of platelet adhesion, platelet spreading, and
platelet thrombus formation (ie, platelet-platelet interactions) as
sequential processes, for at least some platelets (follower platelets),
tethering through filopodia can precede adhesion to collagen and
spreading over collagen or vanguard platelets. Release of granule
contents from vanguard platelets could facilitate this process by
providing high local concentrations of platelet activating agents (eg,
adenosine diphosphate [ADP], thromboxane A2, serotonin)
and adhesive glycoproteins (including fibrinogen and von Willebrand
factor) that facilitate platelet-platelet
interactions.30,31 Release of adhesive glycoproteins could
also decorate nearby collagen, thus supporting GPIIb/IIIa-dependent
platelet spreading. We propose that the unique thrombogenicity of
collagen-coated surfaces results from the ability of one or more of the
collagen receptors, probably primarily Quantitative analysis of the patterns of initial platelet
deposition with untreated human and wild-type mouse platelets
demonstrated that they conformed to that predicted by a Poisson
distribution, indicating that the adhesion events occurred randomly.
Thus, so-called platelet recruitment is not an active process of
diverting platelets from where they would otherwise land randomly;
rather, the ability to support stable platelet-platelet interactions
allows the platelets to deposit randomly, even when that means landing
on top of another platelet (Figure 7).
Ultimately, the platelets develop into a sheetlike multilayered mass in
which individual platelet borders are difficult to discern and in which
there is active movement of platelet organelles and ruffling of
platelet membranes.
Studies with human platelets from a patient with Glanzmann
thrombasthenia and human platelets in the presence of antibody 7E3 or
tirofiban, as well as with wild-type mouse platelets in the presence of
antibody 1B5 or platelets from mice deficient in There are 2 major limitations to our study. First, we investigated
adhesion and thrombus formation under static conditions at 22°C and
did not use whole blood. Thus, it is possible that our observations are
not relevant to in vivo conditions under flow. However, we and others
have observed islands of platelet thrombi when platelets (in either
buffer or whole blood) interact with a collagen-coated surface under
flow in a parallel plate chamber at 37°C.19,32-35
Moreover, our finding of decreased platelet spreading on collagen in
the absence of Our studies also have potential significance for understanding the mechanism of action of abciximab, the Fab fragment of the murine/human chimeric derivative of antibody 7E3, which is used to prevent ischemic complications of percutaneous coronary interventions, and tirofiban. Thus, though these agents do not formally prevent platelet adhesion to collagen, they prevent platelet thrombus formation and alter the pattern of platelet deposition, decrease platelet spreading, and prevent the development of sheetlike masses of platelets.
We thank Dr Ronald Gordon and Norman Katz for their assistance with the scanning electron microscopy studies and Dr Scott Henderson and Valerie Williams for their assistance with the transmission electron microscopy studies, which were performed in the Mount Sinai School of Medicine Microscopy Center.
Submitted March 26, 2002; accepted September 9, 2002.
Supported by grant 19278 from the National Heart, Lung and Blood Institute and supported in part by General Clinical Research Center grant M01-RR00102 from the National Center for Research Resources at the National Institutes of Health.
B.S.C. is an inventor of abciximab and, in accordance with Federal law and the policies of the Research Foundation of the State University of New York, shares in royalties paid to the Foundation for sales of abciximab.
The online version of the 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: Barry S. Coller, Laboratory of Blood and Vascular Biology, The Rockefeller University, 1230 York Ave, New York, NY 10021; e-mail: collerb{at}rockefeller.edu.
1. Parise LV, Smyth SS, Coller BS. Platelet morphology, biochemistry and function. In: Beutler E,Lichtman MA,Coller BS,Kipps TJ,Seligsohn U, eds. Williams Hematology. New York, NY: McGraw-Hill; 2000:1357-1408.
2.
Wu YP, de Groot PG, Sixma JJ.
Shear-stress-induced detachment of blood platelets from various surfaces.
Arterioscler Thromb Vasc Biol.
1997;17:3202-3207 3. Polanowska-Grabowska R, Simon CG Jr, Gear AR. Platelet adhesion to collagen type 1, collagen type 1V, von Willebrand factor, fibronectin, laminin and fibrinogen: rapid kinetics under shear. Thromb Haemost. 1999;81:118-123[Medline] [Order article via Infotrieve].
4.
Badimon L, Badimon JJ, Turitto VT, Vallabhajosula S, Fuster V.
Platelet thrombus formation on collagen type 1: a model of deep vessel injury: influence of blood rheology, von Willebrand factor, and blood coagulation.
Circulation.
1988;78:1431-1442
5.
Kirchhofer D, Tschopp TB, Steiner B, Baumgartner HR.
Role of collagen-adherent platelets in mediating fibrin formation in flowing whole blood.
Blood.
1995;86:3815-3822
6.
Bastida E, Escolar G, Ordinas A, Sixma JJ.
Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces.
Blood.
1987;70:1437-1442
7.
Moog S, Mangin P, Lenain N, et al.
Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation.
Blood.
2001;98:1038-1046 8. Sakariassen KS, Muggli R, Baumgartner HR. Measurements of platelet interaction with components of the vessel wall in flowing blood. Methods Enzymol. 1989;169:37-70[Medline] [Order article via Infotrieve].
9.
Coller BS, Beer JH, Scudder LE, Steinberg MH.
Collagen-platelet interactions: evidence for a direct interaction of collagen with platelet GPIa/IIa and an indirect interaction with platelet GPIIb/IIa mediated by adhesive proteins.
Blood.
1989;74:182-192
10.
Diquelou A, Lemozy S, Dupouy D, Boneu B, Sakariassen K, Cadroy Y.
Effect of blood flow on thrombin generation is dependent on the nature of the thrombogenic surface.
Blood.
1994;84:2206-2213
11.
Saelman EU, Kehrel B, Hese KM, de Groot PG, Sixma JJ, Nieuwenhuis HK.
Platelet adhesion to collagen and endothelial cell matrix under flow conditions is not dependent on platelet glycoprotein IV.
Blood.
1994;83:3240-3244
12.
Saelman EU, Nieuwenhuis HK, Hese KM, et al.
Platelet adhesion to collagen types I through VIII under conditions of stasis and flow is mediated by GPIa/IIa (
13.
de Groot PG, Sixma JJ.
Role of glycoprotein IIb:IIIa in the adhesion of platelets to collagen under flow conditions [letter].
Blood.
1997;89:1837 14. Sixma JJ, van Zanten GH, Huizinga EG, et al. Platelet adhesion to collagen: an update. Thromb Haemost. 1997;78:434-438[Medline] [Order article via Infotrieve].
15.
Verkleij MW, Morton LF, Knight CG, de Groot PG, Barnes MJ, Sixma JJ.
Simple collagenlike peptides support platelet adhesion under static but not under flow conditions: interaction via alpha2 beta1 and von Willebrand factor with specific sequences in native collagen is a requirement to resist shear forces.
Blood.
1998;91:3808-3816
16.
Dickeson SK, Mathis NL, Rahman M, Bergelson JM, Santoro SA.
Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.
J Biol Chem.
1999;274:32182-32191 17. Clemetson KJ. Platelet collagen receptors: a new target for inhibition? Haemostasis. 1999;29:16-26[CrossRef][Medline] [Order article via Infotrieve].
18.
Savage B, Ginsberg MH, Ruggeri ZM.
Influence of fibrillar collagen structure on the mechanisms of platelet thrombus formation under flow.
Blood.
1999;94:2704-2715
19.
Monnet E, Fauvel-Lafeve F.
A new platelet receptor specific to type III collagen: type III collagen-binding protein.
J Biol Chem.
2000;275:10912-10917 20. Watson S, Berlanga O, Best D, Frampton J. Update on collagen receptor interactions in platelets: is the two-state model still valid? Platelets. 2000;11:252-258[CrossRef][Medline] [Order article via Infotrieve]. 21. Moroi M, Onitsuka I, Imaizumi T, Jung SM. Involvement of activated integrin alpha2beta1 in the firm adhesion of platelets onto a surface of immobilized collagen under flow conditions. Thromb Haemost. 2000;83:769-776[Medline] [Order article via Infotrieve].
22.
Emsley J, Knight CG, Farndale RW, Barnes MJ, Liddington RC.
Structural basis of collagen recognition by integrin
23.
Lengweiler S, Smyth SS, Jirouskova M, et al.
Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa ( 24. Hodivala-Dilke KM, Tsakiris DA, Rayburn H, et al. Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J Clin Invest. 1999;103:229-238[Medline] [Order article via Infotrieve]. 25. Coller BS. A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet GPIIb/IIIa complex. J Clin Invest. 1985;76:101-108[Medline] [Order article via Infotrieve].
26.
Smyth SS, Reis ED, Vaananen H, Zhang W, Coller BS.
Variable protection of 27. Seligsohn U, Coller BS, Zivelin A, Plow EF, Ginsberg MH. Immunoblot analysis of platelet GPIIb in patients with Glanzmann thrombasthenia in Israel. Br J Haematol. 1989;72:415-423[Medline] [Order article via Infotrieve].
28.
Coller BS, Seligsohn U, Little PA.
"Type 1" Glanzmann thrombasthenia patients from the Iraqi-Jewish and Arab populations in Israel can be differentiated by platelet glycoprotein IIIa immunoblot analysis.
Blood.
1987;69:1696-1703
29.
Coller BS, Cheresh DA, Asch E, Seligsohn U.
Platelet vitronectin receptor expression differentiates Iraqi-Jewish from Arab Patients with Glanzmann thrombasthenia in Israel.
Blood.
1991;77:75-83
30.
Nakamura T, Jamieson GA, Okuma M, Kambayashi J, Tandon NN.
Platelet adhesion to type 1 collagen fibrils: role of GPVI in divalent cation-dependent and -independent adhesion and thromboxane A2 generation.
J Biol Chem.
1998;273:4338-4344
31.
Nakamura T, Kambayashi J, Okuma M, Tandon NN.
Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both
32.
Alevriadou BR, Moake JL, Turner NA, et al.
Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets.
Blood.
1993;81:1263-1276
33.
Turner NA, Moake JL, Kamat SG, et al.
Comparative real-time effects on platelet adhesion and aggregation under flowing conditions of in vivo aspirin, heparin, and monoclonal antibody fragment against glycoprotein IIb-IIIa.
Circulation.
1995;91:1354-1362
34.
Moroi M, Jung SM, Nomura S, Sekiguchi S, Ordinas A, Diaz-Ricart M.
Analysis of the involvement of the von Willebrand factor-glycoprotein Ib interaction in platelet adhesion to a collagen-coated surface under flow conditions.
Blood.
1997;90:4413-4424 35. Scudder LE, Coller BS. Effects of flow on thromboerythrocyte-platelet interactions [abstract]. Blood. 1993;82:161. 36. Weiss HJ, Turitto VT, Baumgartner HR. Further evidence that glycoprotein IIb-IIIa mediates platelet spreading on subendothelium. Thromb Haemost. 1991;65:202-205[Medline] [Order article via Infotrieve]. 37. Sakariassen KS, Nievelstein PFEM, Coller BS, Sixma JJ. The role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendothelium. Br J Haematol. 1986;63:681-691[Medline] [Order article via Infotrieve].
© 2003 by The American Society of Hematology.
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  R. Blue, M. Murcia, C. Karan, M. Jirouskova, and B. S. Coller Application of high-throughput screening to identify a novel {alpha}IIb-specific small- molecule inhibitor of {alpha}IIb{beta}3-mediated platelet interaction with fibrinogen Blood, February 1, 2008; 111(3): 1248 - 1256. [Abstract] [Full Text] [PDF]
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M. Jirouskova, J. K. Jaiswal, and B. S. Coller Ligand density dramatically affects integrin {alpha}IIb{beta}3-mediated platelet signaling and spreading Blood, June 15, 2007; 109(12): 5260 - 5269. [Abstract] [Full Text] [PDF]
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G. R. Van de Walle, A. Schoolmeester, B. F. Iserbyt, J. M. E. M. Cosemans, J. W. M. Heemskerk, M. F. Hoylaerts, A. Nurden, K. Vanhoorelbeke, and H. Deckmyn Activation of {alpha}IIb{beta}3 is a sufficient but also an imperative prerequisite for activation of {alpha}2{beta}1 on platelets Blood, January 15, 2007; 109(2): 595 - 602. [Abstract] [Full Text] [PDF]
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 M. J. E. Kuijpers, V. Schulte, C. Oury, T. Lindhout, J. Broers, M. F. Hoylaerts, B. Nieswandt, and J. W. M. Heemskerk Facilitating roles of murine platelet glycoprotein Ib and {alpha}IIb{beta}3 in phosphatidylserine exposure during vWF-collagen-induced thrombus formation J. Physiol., July 15, 2004; 558(2): 403 - 415. [Abstract] [Full Text] [PDF]
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ková,
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Abstract
Introduction
IIb
3) + 
