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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Theodor Kocher Institute, University of Berne,
Berne, Switzerland; and the Klinik und Poliklinik
für Anästhesiologie und Operative Intensivmedizin,
Experimental and Clinical Haemostasis, University of Münster,
Germany.
The snake venom C-type lectin alboaggregin A (or 50-kd
alboaggregin) from Trimeresurus albolabris was
previously shown to be a platelet glycoprotein (GP) Ib agonist.
However, investigations of the signal transduction induced in platelets
showed patterns of tyrosine phosphorylation that were different from
those of other GPIb agonists and suggested the presence of an
additional receptor. In this study, the binding of biotinylated
alboaggregin A to platelet lysates, as well as affinity chromatography
evaluations of platelet lysates on an alboaggregin A-coated column,
indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this
finding. These experiments also showed that both GPIb and GPVI have a
role in the combined signaling and that the overall direction this
takes can be influenced by inhibitors of one or the other receptor pathway.
(Blood. 2001;97:929-936) Snake venoms contain many components that play a
role in incapacitating or killing prey. Whereas venoms from some snake
families contain mainly neurotoxins, others, such as those from the
Viperidae and Crotalidae families, have principally hemorrhagic
effects.1,2 Among the components that affect hemostasis
are those that act on coagulation factors, platelets, and endothelial
cells. These proteins are divided into several classes, with a major
class being the proteases,3,4 which can activate
coagulation factors or platelets or affect fibrinogen by cleavage of
specific sequences or receptors. Another major class is the
disintegrins,5,6 which function by binding to and blocking
integrins, major adhesion receptors on platelets, and other cells. Many
of these contain RGD (Arg-Gly-Asp) sequences, but other active
sequences have also been found. Snake metalloproteases include
hemorrhagins,7 which contain a zinc-binding sequence
(HEXXH) that can destroy the basal membrane in vessel walls. Domains of
disintegrins and metalloproteases are combined in the ADAMS class of
proteins,5,6 which function by binding to and cleaving
receptors. Several members of this class are known to affect
collagen-platelet interactions by cleaving receptors or binding to collagen.
Another family of proteins that has been investigated extensively is
the snake C-type lectins,8,9 which were given this name
because of the folding structure they contain, thus resembling members
of the C-type lectin family, which are calcium-dependent (hence, the
"C") and bind to sugars (hence, "lectin"). During the past
decade, several snake C-type lectins that interact with coagulation
factors10 as well as with platelet receptors have been
described. Many, such as echicetin, jararaca glycoprotein (GP) Ib BP,
tokaracetin, agkicetin, CHH-A, and CHH-B, were found to interact with
platelet GPIb as inhibitors,9,11-14 whereas others, such
as alboaggregin B, mamushigin, and flavocetins A and
B,15,16 are activators. Still others, such as convulxin, were shown to interact with the collagen receptor
GPVI17,18 or are thought to act by means of the integrin
collagen receptor Alboaggregins A, B, and C are C-type lectins from
Trimeresurus albolabris, the white-lipped tree viper, that
were shown to bind to platelet GPIb.14,15,22 Alboaggregin
B has a simple heterodimeric structure23 and agglutinates
platelets by binding to GPIb but does not cause major activation.
Alboaggregin A is tetrameric and activates platelets strongly,
presumably by means of a clustering mechanism. Both alboaggregins
agglutinate fixed platelets by cross-linking receptors between
platelets. The primary structure of alboaggregin A, consisting of a
sequence of 4 subunits, has been determined.22 Studies of
the signaling in platelets induced by alboaggregin A have suggested
that it represents signaling by means of GPIb.14,22,24
Because alboaggregin A gives much stronger signals than other GPIb
ligands, it was proposed that it cross-links to a higher degree than
the other agonists and could therefore be used to explore GPIb
signaling pathways that activate platelets.24 However,
because the pattern of tyrosine phosphorylation induced in platelets by
alboaggregin A differs considerably from that caused by other ligands
of GPIb, an alternative explanation for its strong agonist effects on
platelets could be that it binds not only to GPIb but also to another
platelet receptor. In this study, we demonstrated that this alternative explanation is correct: we found evidence that, along with binding to
GPIb, alboaggregin A binds to platelet GPVI and that this is the major
receptor involved in platelet activation by this snake C-type lectin.
Materials
The monoclonal antibody (mAb) anti-GPIb Echicetin was purified from lyophilized Echis carinatus
sochureki venom (Latoxan, Rosans, France)9,27 and
convulxin was purified from lyophilized Crotalus durissus
terrificus venom (Sigma, St Louis, MO)17 as described
previously. Lyophilized trimeresurus (T) albolabris venom
was from Latoxan. Polyvinylidene difluoride (PVDF) membranes
(PolyScreen) were from DuPont NEN.
Octanoyl-N-methylglucamide (ONMG) and
nonanoyl-N-methylglucamide (NNMG) were from Oxyl Chemie (Bobingen, Germany).
Purification of alboaggregin A
Preparation of platelets for gel filtration Venous blood from healthy adult volunteers who had not taken any medication that affects platelet function for at least 2 weeks before the study was obtained by venipuncture of the antecubital vein. Blood was anticoagulated with trisodium citrate (9 parts blood and 1 part trisodium citrate [0.108 M]). Platelet-rich plasma (PRP) was prepared by centrifugation at 250g for 10 minutes at room temperature. Platelets were gel filtered on Sephadex CL-2B equilibrated in buffer A (127 mM NaCl, 2.7 mM potassium chloride [KCl], 0.42 mM sodium phosphate, monobasic [NaH2PO4], 12 mM sodium bicarbonate, 1 mM magnesium chloride, 5.5 mM glucose, and 3.5% BSA [pH 7.35]) containing 0.1 U/mL apyrase.28Platelet washing, aggregation, fixation, phosphorylation times, and immunoprecipitation Human platelets were isolated from buffy coats less than 20 hours after blood collection (Central Laboratory, Swiss Red Cross Blood Transfusion Service). To each buffy coat, 30 mL of 100 mM citrate (pH 6.5) was added. PRP and the platelet pellet were isolated by successive centrifugation steps. Platelets were resuspended in buffer B (113 mM NaCl; 4.3 mM potassium phosphate, dibasic; 4.3 mM sodium phosphate, dibasic, 4.4 mM NaH2PO4, and 5.5 mM glucose [pH 6.5]) and centrifuged at 250g for 5 minutes. The platelet-rich supernatant was centrifuged at 1000g for 10 minutes, and platelets were again washed with buffer B. Washed platelets were resuspended in buffer C (20 mM HEPES, 140 mM NaCl, 4 mM KCl, and 5.5 mM glucose [pH 7.4]) and the platelet count was adjusted to 5 × 108 platelets/mL by dilution with buffer C. Samples were kept at room temperature until used for aggregation studies.Platelet aggregation was monitored by assessing light transmission in an aggregometer (Lumitec), with continuous stirring at 1100 rpm at 37°C. Platelets were preincubated in buffer containing 2 mM calcium chloride (CaCl2) at 37°C for 2 minutes before the measurement was started by adding the samples for analysis. Platelets were fixed by incubation with 0.5% formaldehyde for 0.5 hour, followed by washing twice with phosphate-buffered saline (PBS; pH 7.4). For the tyrosine phosphorylation time-range experiments, aliquots (700 µL; 5 × 108 platelets/mL) of control, resting platelets, and activated platelets at appropriate time points, were solubilized in PBS containing 1% sodium dodecyl sulfate (SDS) with 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EDTA, 2 mM N-ethyl-maleimide (NEM), and 2 mM sodium orthovanadate. For immunoprecipitation, aliquots (700 mL; 5 × 108 platelets/mL) of control, resting, and activated platelets were solubilized in PBS containing 1.2% Triton X-100 with 1 mM PMSF, 5 mM EDTA, 2 mM NEM, and 2 mM sodium orthovanadate. After centrifugation, platelet lysates, which were precleared with protein A-Sepharose, were stirred for 2 hours with specific antibodies before 20 µL protein A-Sepharose was added. The lysates were then incubated for 6 to 8 hours. Alboaggregin A-agarose and biotinylation of alboaggregin A FPLC-purified alboaggregin A (500 µg) was dialyzed against PBS (pH 7.8). N-hydroxysuccinimidyl-activated agarose (500 µL) was washed 3 times in PBS (pH 7.8) and incubated with dialyzed alboaggregin A overnight at 4°C. Reactive groups were blocked with 200 mM glycine (2 hours at 4°C). For biotinylation, alboaggregin A dialyzed against PBS (pH 8) was incubated with BcapNHS (20 µg/mg alboaggregin A) for 1 hour at room temperature. Free BcapNHS was removed by Sephadex G-10 gel filtration.Biotinylation of platelets, preparation of Triton X-100 platelet lysate, WGA affinity chromatography, and alboaggregin A affinity chromatography Human platelets were isolated from 16 buffy coats as described above in the presence of 10 µM Iloprost and 5 mM EDTA. Washed platelets were diluted with PBS to 5 × 109/mL and incubated with 100 µg BcapNHS for 1 hour at room temperature. Free BcapNHS was separated by washing 3 times with PBS (pH 6.8). Biotinylated platelets were solubilized in PBS containing 1.2% Triton X-100 with 1 mM PMSF, 5 mM EDTA, and 2 mM NEM2. After centrifugation (40 000g for 1 hour at 4°C), the supernatant was applied to a column of WGA-Sepharose 4B equlibrated with buffer D (10 mM Tris, 130 mM NaCl, and 2.5 mM EDTA [pH 7.4]). The column was washed thoroughly with buffer D containing 0.2% ONMG. The bound material was eluted with 2.5% N-acetylglucosamine in buffer E (10 mM Tris, 30 mM NaCl, and 2.5 mM EDTA [pH 7.4]) containing 0.2% ONMG. Fractions containing membrane glycoproteins were pooled and loaded on an affinity chromatography column coated with alboaggregin A and equilibrated with buffer D. The column was washed thoroughly with buffer D containing 0.2% ONMG and then with buffer E containing 0.2% NNMG. The bound material was eluted with 0.08% SDS in buffer E. Fractions containing membrane glycoproteins that bound to alboaggregin A were analyzed by electrophoresis.Flow cytometry Samples were analyzed by using a fluorescence-activated cell-sorter flow cytometer (FACScan; Becton Dickinson, Heidelberg). Excitation was with an argon laser at 488 nm. The FACScan was used in a standard configuration with a 530-nm bandpass filter. Platelets were gated and data were obtained from fluorescence channels in a logarithmic mode. A total of 5000 events were analyzed for each data point.Alboaggregin A-induced procoagulant activity and
CD62P expression was measured as a marker of Alboaggregin A binding to fixed platelets and its inhibition Aliquots of formaldehyde-fixed platelets (100 µL; 2.5 × 107 platelets/mL in PBS [pH 7.4]) were preincubated for 5 minutes with increasing amounts of inhibitors and then incubated with biotinylated alboaggregin A (0.5 µg/mL) for 20 minutes. Subsequently, platelets were washed and incubated with streptavidin-PE for 30 minutes. The platelets were then washed, resuspended in PBS, and analyzed by flow cytometry. Unspecific streptavidin-PE binding to platelets was subtracted. Saturation binding of biotinylated alboaggregin was determined in a separate experiment (data not shown).
Purification of alboaggregin A Alboaggregin A was purified from lyophilized crude T albolabris venom by ion-exchange FPLC using a Fractogel EMD TMAE-650 (S) column and a Q2 column. The final product showed a broad, 50-kd band under nonreducing conditions and 3 bands, at 14, 15, and 16 kd, under reducing conditions, when analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and silver staining (Figure 1). The 14-kd band was noticeably stronger than those at 15 and 16 kd and contained both subunits.
The apparent molecular masses of subunits were comparable with those
reported previously.9,22 After reduction and alkylation,
the 4 subunits of alboaggregin A were separated by high-performance
liquid chromatography (HPLC), and N-terminal sequences were obtained.
The N-terminal amino acid sequences of alboaggregin A subunits were the
same as those described previously.22
Effects of anti-GPIb Abs, echicetin, and convulxin on alboaggregin A binding to fixed platelets Alboaggregin A binding to formaldehyde-fixed platelets was inhibited by anti-GPIb mAb SZ2, polyclonal anti-GPIb Ab, the C-type lectin echicetin (a GPIb-binding protein), and the C-type lectin
convulxin (a GPVI-binding protein) (Figure
2). Formaldehyde-fixed platelets were
incubated with increasing amounts of Abs (20-100 µg/mL) or C-type
lectins (1.6-140 µg/mL) for 5 minutes before the biotinylated
alboaggregin A (0.5 µg/mL) was added. The inhibitory effect of
convulxin, directed against GPVI, was much stronger than the effect of
the inhibitors directed against GPIb. Saturation binding of
biotinylated alboaggregin A and inhibition of biotinylated alboaggregin
A binding by unlabeled alboaggregin A were determined in separate
experiments (data not shown).
Alboaggregin A-induced agglutination and aggregation Alboaggregin A agglutinated formaldehyde-fixed platelets in the absence of any cofactor (Figure 3, lane 1). Maximum agglutination was reached at 1 µg/mL. Additional aggregation experiments were done with unfixed platelets. To evaluate the role of GPIb and GPVI in alboaggregin A-induced aggregation, echicetin was used to inhibit GPIb and Fab fragments of GPVI Ab were used to inhibit GPVI. Thrombin and collagen, respectively, were used as agonists to control the efficiency of inhibition by these agents. Echicetin (15 µg/mL), a GPIb-binding protein, partly blocked alboaggregin A-induced (0.5 µg/mL) and thrombin-induced (0.05 U/mL) aggregation of washed platelets (Figure 3, lane 2 and lane 3). Fab fragments of anti-GPVI Abs partly blocked alboaggregin A-induced (0.5 µg/mL) platelet aggregation and completely blocked collagen-induced (0.36 µg/mL) aggregation (Figure 3, lane 4 and lane 5).
Binding of biotinylated alboaggregin A to platelet lysate Proteins in platelet lysate were separated by SDS-PAGE (7%-17% acrylamide gradient) and electroblotted on PVDF membranes. The membranes were incubated with biotinylated alboaggregin A and avidin-coupled alkaline phosphatase or with anti-GPVI Ab and antirabbit IgG-coupled alkaline phosphatase. Both were stained with BCIP and NBT. Under nonreduced conditions, biotinylated alboaggregin A bound to a band with the same molecular mass as GPVI (Figure 4B). Unspecific binding of avidin phosphatase to platelet lysate could be excluded (Figure 4C). Figure 4D, lane 3, clearly shows binding of alboaggregin A to GPVI immunoprecipitated with anti-GPVI antibodies and to GPVI purified on a convulxin-coated column (lane 4). Figure 4D, lanes 1 and 2, shows immunoprecipitations with the preimmune serum of the anti-GPVI Ab and an anti-GPV Ab as controls.
Binding of biotinylated platelets to alboaggregin A The fractions enriched in biotinylated platelet glycoproteins from WGA column chromatography were loaded on an alboaggregin A-agarose affinity column. Fractions eluted with increasing amounts of SDS were analyzed by using SDS-PAGE (7%-17% acrylamide gradient) and transferred to PVDF membranes. The membranes were then incubated with avidin-coupled alkaline phosphatase or with anti-GPVI, anti-GPIb, anti-GPIb , or anti-GPIX Abs and
peroxidase-conjugated antirabbit Abs. Membranes were stained with BCIP
and NBT or bands detected with chemiluminescence. Fractions that eluted
with 0.08% SDS contained GPIb, GPIb, GPIX, and GPVI
(Figure 5).
Alboaggregin A-induced platelet procoagulant activity
and , which inhibits thrombin binding but not von
Willebrand factor (vWf) binding, and mAb SZ2, which prevents both
thrombin and vWf binding to GPIb, inhibited the procoagulant
response of platelets to low but not to high doses of alboaggregin A
(Figure 6). The antibodies were used at 20 µg/mL and were incubated
with gel-filtered platelets for 10 minutes before treatment with
alboaggregin A (0.1-2.5 µg/mL). Blockage of GPVI with Fab fragments
of anti-GPVI Abs or blockage of GPIb with anti-GPIb Abs inhibited
procoagulant responses to low doses of alboaggregin A (Figure 6A). The
optimal antibody concentrations were determined
separately.
Granule release from gel-filtered platelets treated with alboaggregin A (0.1-5 µg/mL) and detected with FITC-labeled anti-CD62P Ab is shown in Figure 6B. Inhibition of GPIb or GPVI clearly reduced granule release. The concentrations of antibodies used were those mentioned above. Alboaggregin A-induced tyrosine
phosphorylation, inhibitory effects of Fab fragments of anti-GPVI Ab,
and immunoprecipitation of Fc  2, Syk,
Lyn, Fyn, and FcR, showed raised levels of tyrosine phosphorylation
after activation with alboaggregin A. Fc, LAT, and PLC2 were
clearly tyrosine phosphorylated after platelet activation with
alboaggregin A at either 1 minute (Fc), 2 minutes (PLC2), or 30 seconds (LAT), whereas resting platelets had no or very low levels of
tyrosine phosphorylation. Figure 7 shows that tyrosine phosphorylation of several proteins during activation with alboaggregin A was inhibited
by Fab fragments of anti-GPVI Abs. Tyrosine phosphorylation of LAT was
delayed for more than 2 minutes and tyrosine phosphorylation of Fc
was inhibited completely under these conditions, indicating that Fc
phosphorylation by alboaggregin A occurs mainly by means of activation
of GPVI. In Figure 7B, 7C, and 7D, the upper bands show the level of
tyrosine phosphorylation in immunoprecipitates from resting and
alboaggregin A-activated platelets with anti-Fc, anti-LAT, and
anti-PLC2 Abs, respectively. The lower bands show the same blots
after stripping and reblotting with the corresponding antibodies to
confirm that equal amounts of each component were immunoprecipitated
from each platelet preparation.
Tyrosine phosphorylation signal transduction by alboaggregin A and the inhibitory effect of echicetin Figure 8 shows a time range of tyrosine phosphorylation for platelets activated with 0.3 µg/mL alboaggregin A compared with platelets that were preincubated with 15 µg/mL echicetin. In the presence of echicetin or SZ2 (data not shown), the overall pattern of tyrosine phosphorylation was somewhat reduced but mainly was slower; however, phosphorylation of certain proteins, notably Fc and LAT, increased.
Because the signaling induced in platelets by the C-type
lectin alboaggregin A did not correspond to that expected for a simple GPIb agonist, the specificity of alboaggregin A for platelet receptors was examined in detail by using several approaches. Alboaggregin A
(50-kd alboaggregin) was purified from lyophilized T
albolabris venom, essentially by using previously described
methods,14,22 as a single, 50-kd nonreduced band and as 3 bands at 14, 15, and 16 kd in the reduced protein (Figure 1). The
reduced band at 14 kd stained more strongly than the other 2 and
contained both the Alboaggregin A was labeled with biotin and used in a ligand blotting
experiment with platelet lysate separated by SDS-PAGE and blotted on
PDVF membrane. The biotinylated alboaggregin A detected specifically a
broad band at about 60 kd on the blot from the nonreduced lane but did
not detect any band specifically on the blot from the reduced lane
(Figure 4B). Polyclonal antibodies to GPVI detected bands at 60 kd and
65 kd, respectively, in lanes run in parallel. The biotinylated
alboaggregin A also detected a 60-kd band in immunoprecipitates with an
anti-GPVI antibody from platelet lysates and in bound material from a
convulxin-agarose affinity column (Figure 4). Biotinylated alboaggregin
A detected GPVI but failed to detect GPIb, probably because the
conformation of GPVI was maintained by its disulfide bridges, thus
allowing renaturing on the blot, whereas for GPIb Alboaggregin A was coupled to agarose and used for affinity
chromatography assessments of nonionic detergent-solubilized platelet lysate. After extensive washing, the bound material was eluted with
SDS. Both it and the last washes were analyzed by SDS-PAGE. Western blotting was also done with antibodies to GPIb Induction of platelet procoagulant activity in response to
alboaggregin A was examined by using binding of annexin V to
surface-exposed, negatively charged phospholipids or by measuring the
generation of thrombin with a chromogenic assay.29 These
studies clearly showed that alboaggregin A could induce this effect and
was only slightly affected by antibodies against GPIb (Figure 6).
Agonists specific for GPVI were previously shown to be efficient
inducers of platelet procoagulant activity,30,31 whereas
GPIb has been only marginally implicated in this
function32 or in response to thrombin as the specific
agonist.29 The tyrosine signal-transduction pathways of
platelets treated with alboaggregin A were previously investigated by
several groups14,24 and found to resemble closely those
induced by collagen or convulxin.17,33 Thus, Fc The effects of inhibitors of either GPIb or GPVI on tyrosine
phosphorylation induced by alboaggregin A were therefore examined. Fab
fragments of polyclonal anti-GPVI had a strong inhibitory effect on
overall tyrosine phosphorylation, although some bands, particularly
those at 60 and 64 kd, still showed a strong increase, whereas other
bands (in the range of 30-40 kd and 100-120 kd) showed a weaker
increase and were later than in the control platelets (Figure 7).
Tyrosine phosphorylation of Fc These findings suggest that the structure of alboaggregin
A,22 together with its binding properties, is an
The question remains why the use of GPIb as a coreceptor is
advantageous for the snake. Several explanations appear possible. One
is that the larger number of GPIb molecules per platelet (at least
25 000 and probably 50 000) compared with GPVI molecules (2000-5000)
permits a more efficient use of alboaggregin A in activating platelets
and organizing links between platelets. Alternatively (or in addition),
cross-linking GPIb and GPVI may amplify the signaling from each by
means of a cross-talk mechanism. The results described here support
both possibilities and indicate that it is not only GPVI that provides
a signal. At lower concentrations of alboaggregin A, signaling by means
of GPIb in addition to GPVI permits a strong activation of platelet
tyrosine phosphorylation while shifting the mechanism away from the
Fc
We thank Dr J.-P. Kinet, Dr A.V. Mazurov, and Dr S. Santoso, respectively, for the Fc
Submitted June 1, 2000; accepted October 11, 2000.
Work at the Theodor Kocher Institute was supported by grant 31-52396.97 from the Swiss National Science Foundation to K.J.C.
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: K.J. Clemetson, Theodor Kocher Institute, University of Berne, Freiestrasse 1, CH-3012 Berne, Switzerland; e-mail: clemetson{at}tki.unibe.ch.
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A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase C
© 2001 by The American Society of Hematology.
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  G. Pula, D. Crosby, J. Baker, and A. W. Poole Functional Interaction of Protein Kinase C{alpha} with the Tyrosine Kinases Syk and Src in Human Platelets J. Biol. Chem., February 25, 2005; 280(8): 7194 - 7205. [Abstract] [Full Text] [PDF]
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J.-R. Nofer, G. Herminghaus, M. Brodde, E. Morgenstern, S. Rust, T. Engel, U. Seedorf, G. Assmann, H. Bluethmann, and B. E. Kehrel Impaired Platelet Activation in Familial High Density Lipoprotein Deficiency (Tangier Disease) J. Biol. Chem., August 6, 2004; 279(32): 34032 - 34037. [Abstract] [Full Text] [PDF]
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S. Kanaji, T. Kanaji, K. Furihata, K. Kato, J. L. Ware, and T. J. Kunicki Convulxin Binds to Native, Human Glycoprotein Ib{alpha} J. Biol. Chem., October 10, 2003; 278(41): 39452 - 39460. [Abstract] [Full Text] [PDF]
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 B. Nieswandt and S. P. Watson Platelet-collagen interaction: is GPVI the central receptor? Blood, July 15, 2003; 102(2): 449 - 461. [Abstract] [Full Text] [PDF]
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D. Crosby and A. W. Poole Physical and Functional Interaction between Protein Kinase C {delta} and Fyn Tyrosine Kinase in Human Platelets J. Biol. Chem., June 27, 2003; 278(27): 24533 - 24541. [Abstract] [Full Text] [PDF]
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M. Mekrache, C. Bachelot-Loza, N. Ajzenberg, A. Saci, P. Legendre, and D. Baruch Activation of pp125FAK by type 2B recombinant von Willebrand factor binding to platelet GPIb at a high shear rate occurs independently of {alpha}IIb{beta}3 engagement Blood, June 1, 2003; 101(11): 4363 - 4371. [Abstract] [Full Text] [PDF]
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 S. Massberg, M. Gawaz, S. Gruner, V. Schulte, I. Konrad, D. Zohlnhofer, U. Heinzmann, and B. Nieswandt A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo J. Exp. Med., January 6, 2003; 197(1): 41 - 49. [Abstract] [Full Text] [PDF]
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X.-Y. Du, J. M. Clemetson, A. Navdaev, E. M. Magnenat, T. N. C. Wells, and K. J. Clemetson Ophioluxin, a Convulxin-like C-type Lectin from Ophiophagus hannah (King Cobra) Is a Powerful Platelet Activator via Glycoprotein VI J. Biol. Chem., September 13, 2002; 277(38): 35124 - 35132. [Abstract] [Full Text] [PDF]
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A. Kasirer-Friede, J. Ware, L. Leng, P. Marchese, Z. M. Ruggeri, and S. J. Shattil Lateral Clustering of Platelet GP Ib-IX Complexes Leads to Up-regulation of the Adhesive Function of Integrin alpha IIbbeta 3 J. Biol. Chem., March 29, 2002; 277(14): 11949 - 11956. [Abstract] [Full Text] [PDF]
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D. Crosby and A. W. Poole Interaction of Bruton's Tyrosine Kinase and Protein Kinase Ctheta in Platelets. CROSS-TALK BETWEEN TYROSINE AND SERINE/THREONINE KINASES J. Biol. Chem., March 15, 2002; 277(12): 9958 - 9965. [Abstract] [Full Text] [PDF]
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N. Asazuma, S. J. Marshall, O. Berlanga, D. Snell, A. W. Poole, M. C. Berndt, R. K. Andrews, and S. P. Watson The snake venom toxin alboaggregin-A activates glycoprotein VI Blood, June 15, 2001; 97(12): 3989 - 3991. [Abstract] [Full Text] [PDF]
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W. Bergmeier, D. Bouvard, J. A. Eble, R. Mokhtari-Nejad, V. Schulte, H. Zirngibl, C. Brakebusch, R. Fassler, and B. Nieswandt Rhodocytin (Aggretin) Activates Platelets Lacking alpha 2beta 1 Integrin, Glycoprotein VI, and the Ligand-binding Domain of Glycoprotein Ibalpha J. Biol. Chem., June 29, 2001; 276(27): 25121 - 25126. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
RI
Platelets in the presence of echicetin, 
platelets in the presence
of convulxin, 
platelets in the presence of mAb SZ2, and 
platelets in the presence of polyclonal rabbit anti-GPIb Abs. The
fluorescence of 5000 platelets per measuring point was determined by
flow cytometry. Data are the mean results from 3 experiments using
platelets from different donors.
25 000 platelets/µL in the presence of mAb VM16d, and 
25 000 platelets/µL in the presence of Fab fragments of
anti-GPVI Abs.
cDNA sequence, functional characterization, and three-dimensional modeling.
Thromb Haemost.
2000;83:119-126