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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Physiology, Pharmacology, and
the Sol Sherry Thrombosis Research Center, Temple University School of
Medicine, Philadelphia, PA.
Collagen activates platelets by transducing signals through
glycoprotein VI (GPVI). It is not clear whether collagen can directly activate fibrinogen receptors on the adherent platelets without a role
for positive feedback agonists. We investigated the contribution of
secondary G protein signaling to the mechanism of GPVI-stimulated platelet aggregation using the GPVI-selective agonists, convulxin and
collagen-related peptide (CRP) as well as collagen. Adenosine diphosphate (ADP) scavengers or ADP receptor antagonists shifted the
concentration-response curve slightly to the right at low concentrations of convulxin, whereas platelet aggregation at higher concentrations of convulxin was unaffected by these agents. ADP receptor antagonists shifted the concentration-response curve of
collagen- or CRP-induced platelet aggregation to the right at all the
concentrations. Protein kinase C inhibitor, Ro 31-8220, or a
calcium chelator 5,5'-dimethyl-BAPTA shifted the
concentration-response curve of convulxin-induced platelet aggregation
to the right. In addition, pretreatment with both Ro 31-8220 and
dimethyl-BAPTA resulted in total inhibition of convulxin-mediated
aggregation. Blockade of either the calcium- or protein kinase
C-regulated pathway leads to inhibition of fibrinogen receptor
activation on platelets adherent to collagen, but inhibition of both
pathways leads to abolished fibrinogen receptor activation. We conclude that collagen-induced activation of fibrinogen receptor on adherent platelets through GPVI signaling occurs without any significant role
for secreted ADP or thromboxane A2. Furthermore, protein kinase C- and calcium-regulated pathways independently contribute to
GPVI-mediated platelet aggregation.
(Blood. 2002;99:3228-3234) Contact of circulating platelets with exposed
subendothelium results in the release, generation, or exposure of
agonists, which in turn can activate platelets in a positive feedback
loop. Among these agonists are collagen (exposed), thrombin and
thromboxane A2 (generated), and adenosine diphosphate
(ADP), epinephrine and serotonin (released).1
Agonist-stimulated platelets change shape and subsequently aggregate,
which requires activation of the platelet integrin adhesion receptor
Even though platelets express a number of receptors for collagen, it is
generally accepted that adhesion of platelets to collagen is mainly
mediated by integrin There has been a rapid accumulation of knowledge with respect to the
signaling molecules involved prior to PLC Materials
Isolation of platelets
Analysis of platelet aggregation Agonist-induced platelet aggregation was determined by measuring the transmission of light through a 0.5-mL sample of washed platelets with constant stirring (900 rpm) in a lumiaggregometer at 37°C (Chrono-Log, Havertown, PA). The recorder output speed was set to 0.2 mm/s. The baseline was set using Tyrode solution as a blank. All treatments were performed just prior to the addition of agonist. Platelets were loaded with 20 µM dimethyl-BAPTA by incubating the washed platelets with the acetoxylmethyl ester form or dimethylsulfoxide (control) for 10 minutes. Platelet aggregation in response to different concentrations of agonist was measured in the presence of exogenously added fibrinogen (1 mg/mL) whenever Ro 31-8220 (10 µM) or 5-5'-dimethyl-BAPTA (20 µM) was used. Extent of aggregation was measured 210 seconds after the addition of the agonist. The maximum aggregation extent in the absence of any agent was taken as 100% and the remaining values were normalized to this value. In the case of collagen, extent of aggregation in the presence of SC57101, a fibrinogen receptor antagonist, was taken as 0%. All experiments were repeated at least 3 times using platelets from different donors.Measurement of platelet secretion [3H]5-HT was loaded into platelets by incubating PRP with 1 µCi/mL (37 KBq/mL) for 30 minutes, followed by 15 minutes at room temperature. Platelets were then processed as described above. Imipramine was added to the Tyrode solution at a final concentration of 1 µM during resuspension of the washed platelets to prevent reuptake of secreted [3H]5-HT. The activation of labeled [3H]5-HT platelets was performed in the aggregometer at 37°C with stirring (900 rpm) and was stopped after 2 minutes with the addition of formaldehyde/EDTA. Samples were collected and centrifuged at 5000g for 1 minute, and the radioactivity of the supernatant was measured using Wallac 1409 liquid scintillation counter (Perkin Elmer Life Sciences, Gaithersburg, MD). Platelet secretion was expressed as the percentage of the total [3H]5-HT content.Measurement of cytoplasmic concentrations of ionized Ca++ The PRP was incubated at 37°C with 3 µM Fura PE-3 acetoxymethyl ester for 30 minutes followed by 15 minutes at room temperature. Platelets were then isolated as described above. Aliquots (0.5 mL) of the platelet suspension were stirred (900 rpm) in a water-jacketed cuvette maintained at 37°C during activation. Fluorescence was constantly measured using an Aminco-Bowman Series 2 luminescence spectrometer with settings of 340 nm (excitation) and 510 nm (emission). All experiments were repeated at least 3 times using platelets from different donors.Platelet adhesion to collagen The method of Smith and Dangelmaier27 was used to determine platelet adhesion to collagen. Briefly, PRP was incubated with [3H]-oleic acid (1 µCi/mL; 37 KBq/mL) and 1 mM aspirin at 37°C for 1 hour. Platelets were then isolated as described above. One milliliter samples were stirred at 800 rpm in an aggregometer at 37°C with addition of SC57101 (1 µM), SQ29.548 (100 µM), A3P5P (1 mM), and AR-C66096 (1 µM). Collagen (50 µg/mL) was added and the incubation continued for 2 minutes. Some samples were treated with Ro 31-8220 (10 µM, 5 minutes at 37°C) or dimethyl-BAPTA (20 µM, 10 minutes at 37°C) before collagen treatment. Following incubation, the platelet suspensions were decanted into a manifold containing 10-µm nylon disks under vacuum. The disks were rinsed with 2 mL Tyrode solution and either transferred directly to liquid scintillation cocktail for measurement of adhesion or placed in a 6-well plate for PAC-1 binding measurements.PAC-1 binding to platelets adhered to collagen The 10-µm nylon disks were placed in 6-well plates, each well containing 2 mL Hanks balanced salt solution (HBSS). The disks were blocked with HBSS containing 1% BSA for 45 minutes at room temperature with gentle agitation. The blocking solution was replaced with a PAC-1 antibody (1:1000 dilution in blocking buffer) and agitated for 30 minutes at room temperature. The disks were then washed 3 times in HBSS, blocked for an additional 15 minutes at room temperature, and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000 dilution in blocking buffer) for 30 minutes at room temperature. The disks were washed 3 times and exposed to O-phenylenediamine (OPD) and H2O2. When adequate color change was reached (10 minutes), an equal volume of sample was mixed with 2.5M H2SO4 and the optical density was read at 490 nm. Data are expressed as relative PAC-1 binding (a ratio of the absorbance at 490 nm to the percent of platelets adhered to collagen times 100%).
Effect of ADP scavengers on GPVI-mediated platelet aggregation In the cases of ADP and thromboxane A2, it is known that concomitant signaling through both Gi-coupled and Gq-coupled receptors is required for platelet aggregation.19,28,29 However, the GPVI receptor does not couple directly to either Gi or Gq. Therefore, to determine whether G protein-coupled ADP receptors play any significant role in GPVI-mediated platelet aggregation through secreted agonists, we used ADP-scavenging enzyme systems. Because we used aspirin-treated platelets throughout the study, we have eliminated the generation of thromboxane A2 and its contribution to platelet activation in this system. A concentration-response analysis of convulxin-mediated aggregation revealed that platelet shape change occurred at lower concentrations, whereas higher concentrations caused platelet aggregation (Figure 1). When creatine phosphate (10 mM) and creatine phosphokinase (40 U/mL) were used as ADP scavengers, a significant inhibitory effect was achieved on convulxin-induced platelet aggregation at lower convulxin concentrations (Figure 2A). The concentration-response curve shifted slightly to the right at lower concentrations of convulxin, but the extent of platelet aggregation was unaffected with higher concentrations of convulxin. However, in the case of collagen or CRP, the ADP scavenger system shifted the concentration response curves to the right with all concentrations (Figure 2B,C). This implied that at low concentrations of convulxin and all concentrations of collagen and CRP, platelet aggregation is potentiated by secreted ADP.
Effect of receptor-selective antagonists on GPVI-mediated platelet aggregation To elucidate the role of the specific ADP receptor involved in GPVI-mediated platelet aggregation, we used ADP receptor selective antagonists. The Gq-coupled P2Y1 receptor antagonist A3P5P and the Gi-coupled P2Y12 receptor antagonist AR-C66096 marginally inhibited convulxin-induced platelet aggregation at the low convulxin concentrations (Figure 3A). The Gi-coupled 2A receptor antagonist yohimbine was without
any significant effect (not shown). When both P2 receptor antagonists
were combined, no cumulative effect on convulxin-mediated aggregation
was observed (Figure 3A). When collagen or CRP was used as a GPVI
agonist, either the P2Y1 receptor antagonist (MRS2179) or the P2Y12
receptor antagonist (AR-C69931MX) shifted the concentration-response
curve to the right (Figure 3B,C). In case of collagen, MRS2179 appeared
to be more potent than AR-C69931MX in causing the rightward shift,
whereas both antagonists were equally effective in CRP-induced platelet
aggregation. When both P2 receptor antagonists were combined, the
concentration-response curve for CRP-induced platelet aggregation
shifted further to the right.
Effect of protein kinase C inhibition on GPVI-mediated platelet responses We have previously shown that U46619-induced aggregation is dependent on secretion because the aggregation was completely blocked by the cell-permeable PKC inhibitor Ro 31-8220, a known inhibitor of platelet secretion.30 Therefore, we wanted to determine whether convulxin-induced aggregation was also sensitive to inhibition of secretion by Ro 31-8220. Platelet secretion of dense granule contents is totally abolished by 10 µM Ro 31-8220 in response to different concentrations of convulxin (Figure 4). In the presence of Ro 31-8220, lower concentrations of convulxin only caused shape change and did not induce significant aggregation (Figure 5A). However, when higher concentrations of convulxin were used, the concentration response curve of convulxin-induced platelet aggregation shifted to the right in the presence of Ro 31-8220. Similarly the concentration-response curves for platelet aggregation were shifted to the right with collagen (Figure 5B) in the presence of Ro 31-8220.
Effects of 5,5'-dimethyl-BAPTA on convulxin-induced platelet aggregation We investigated the role of increased cytosolic Ca++ as a possible means by which platelets aggregate in response to convulxin. The normal increase in cytosolic Ca++, which occurs in response to convulxin, does not occur in platelets loaded with the calcium chelator 5,5'-dimethyl-BAPTA (not shown). In the absence of increased cytosolic Ca++, platelet aggregation in response to convulxin or collagen was inhibited significantly (Figure 5). As in the case of Ro 31-8220 treatment, the concentration-dependence of convulxin- or collagen-induced platelet aggregation shifted to the right in the presence of dimethyl-BAPTA.Because blockade of either the PKC-regulated pathway (with Ro 31-8220) or calcium-regulated pathway (with dimethyl-BAPTA treatment) did not abolish GPVI-mediated platelet aggregation, we investigated the effect of inhibiting both the pathways on GPVI-mediated platelet aggregation. In the presence of both Ro 31-8220 and dimethyl-BAPTA, convulxin- or collagen-induced platelet aggregation is abolished (Figure 5). Although platelet aggregation reflects the activation of fibrinogen
receptor, in the case of collagen, platelet aggregation is a reflection
of fibrinogen receptor activation on platelets not adherent to collagen
fibers. To evaluate the role of PKC-regulated and calcium-regulated
pathways in GPVI-induced fibrinogen receptor activation on platelets
adherent to collagen, we established a PAC-1 binding assay. In this
assay, bound PAC-1 was estimated by a colorimetric assay using an
enzyme-linked secondary antibody. The platelets adherent to collagen
were estimated by [3H]-oleic acid. Thus, the ratio of
PAC-1 binding to adherent platelets reflects relative fibrinogen
receptor activation. As seen in Figure 6,
fibrinogen receptor is activated on aspirin-treated platelets adherent
to collagen fibers, even in the presence of ADP receptor antagonists.
This indicated that collagen activates fibrinogen receptor directly on
the adherent platelet without a role for ADP and thromboxane
A2. Furthermore, pretreatment of platelets with Ro 31-8220 or dimethyl-BAPTA resulted in a decrease in fibrinogen receptor
activation on platelets adherent to collagen. The blockade of both
PKC-regulated and calcium-regulated pathways resulted in dramatic
inhibition of fibrinogen receptor activation. These results
indicate that collagen, through GPVI-mediated stimulation of PLC
Upon vascular injury, adhesion of platelets to collagen in the
subendothelium is the first step in platelet activation. Because collagen forms insoluble fibers at physiologic pH and represents a
solid surface, the nature of the collagen-platelet interaction is more
complicated than with other agonists. Activation of collagen-adhered platelets results in generation and secretion of soluble agonists, namely thromboxane A2 and ADP, that further activate
nonadherent platelets.31,32 Therefore, the platelet
aggregation response to low concentrations of collagen that is observed
in an aggregometer is the reflection of aggregation of nonadherent
platelets rather than the collagen-adherent platelets. Thus, the
defective platelet aggregation response to collagen in G Among the many proposed platelet surface receptors for collagen, GPVI has gained credence as the receptor responsible for collagen-induced platelet signal transduction.8,11,35-40 GPVI signaling is rather distinct from that of G protein-coupled receptor agonists in that it goes through a series of tyrosine phosphorylation steps that are not yet fully understood.10,35 Because collagen is insoluble at physiologic pH, it is imperative to use a soluble collagen receptor agonist to delineate the signal transduction events through GPVI on platelets. By virtue of its specific interaction with collagen receptor GPVI, the snake venom protein convulxin15,41,42 or CRP16 is well suited for this task. Because convulxin induces platelet shape change at lower concentrations
and aggregation at higher concentrations (Figure 1), we speculated that
convulxin-induced aggregation requires secretion of granule contents as
is the case for U46619-induced platelet aggregation.20 The
role of secreted ADP on aggregation mediated by convulxin, CRP, or
collagen was examined using receptor-selective antagonists as well as
ADP scavengers. Significant inhibition of aggregation was seen with the
ADP receptor antagonists and ADP scavenger enzymes at low
concentrations of convulxin, whereas little if any effect of the
inhibitors was seen at high convulxin concentrations (Figures 2A and
3A). The Gi-coupled Consistent with the role for PKC activation in platelet secretion,20,30,44 we observed that Ro 31-8220 inhibited convulxin-induced secretion in platelets (Figure 4). The presence of Ro 31-8220 significantly inhibited aggregation even at higher concentrations of convulxin. However, the studies with ADP scavengers and receptor antagonists revealed that convulxin can cause platelet aggregation in the absence of secreted ADP. These results suggested that convulxin-induced platelet aggregation may be dependent on PKC activation. In addition, the inability of Ro 31-8220 to abolish platelet aggregation at high concentrations of convulxin raises the possibility that multiple signaling pathways contribute to convulxin-induced platelet aggregation. Thus, we investigated the contribution of GPVI-induced calcium mobilization on platelet aggregation through the use of the high affinity Ca++ chelator 5,5'-dimethyl-BAPTA. In a profile similar to that seen with Ro 31-8220, the aggregation induced by lower concentrations of convulxin was abolished in the presence of dimethyl-BAPTA, whereas up to 60% inhibition occurred at higher concentrations (Figure 5). Under conditions in which PKC activity and calcium mobilization are simultaneously inhibited by Ro 31-8220 and dimethyl-BAPTA, respectively, GPVI-stimulated platelet aggregation was completely inhibited even at high convulxin concentrations (Figure 5). Similar results were obtained with collagen-induced platelet aggregation (Figure 5B). Fibrinogen receptor activation occurred on platelets adherent to
collagen fibers even in the presence of ADP receptor antagonists (Figure 6). These data confirm the previous studies of Nakamura and
coworkers21,39 through the use of receptor antagonists. When the PKC-regulated pathway or calcium-regulated pathway is blocked,
fibrinogen receptor is still activated on collagen-adherent platelets.
Thus on the adherent platelet, collagen can cause fibrinogen receptor
activation through either the PKC-regulated pathway or the
calcium-regulated pathway. When the PKC-regulate pathway is inhibited
by Ro 31-8220, both phosphorylation events downstream of PKC leading to
In conclusion, our results show that GPVI-stimulated aggregation is mediated independently by 2 different mechanisms, namely the PKC- and calcium-regulated pathways. The role of secreted agonists in response to GPVI-mediated signaling appears to be stimulation of G protein-coupled ADP receptors and the resultant potentiation of the primary aggregation response.
Subsequent to the original submission of this manuscript, Atkinson et al46 showed that Ca++ and protein kinase C, but not release of the secondary agonists ADP and thromboxane A2, are required for full aggregation induced by convulxin, whereas the response induced by collagen shows a much greater dependence on secretion of secondary agonists.
Submitted June 20, 2001; accepted December 14, 2001.
Supported by research grants HL64943 and HL60683 from the National Institutes of Health. T.M.Q. is the recipient of a Postdoctoral Fellowship from the Pennsylvania-Delaware affiliate of the American Heart Association.
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: Satya P. Kunapuli, Temple University Medical School, Department of Physiology, Rm 224 OMS, 3420 N Broad St, Philadelphia, PA 19140; e-mail: kunapuli{at}nimbus.temple.edu.
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Top
Abstract
Introduction
3 to bind fibrinogen and link adjacent
platelets together in an aggregate.
chain is coexpressed with GPVI in platelets and is
phosphorylated on tyrosine residues of its ITAM motif on GPVI-collagen
binding.
) or presence (
) of the ADP scavenger system. The maximum
aggregation extent in the absence of the ADP scavenger system was taken
as 100% and the remaining values were normalized to this value. In the
case of collagen, extent of aggregation in the presence of SC57101, a
fibrinogen receptor antagonist, was taken as zero. Each data point is
the mean ± SE of 3 measurements with different donors.
), or both P2Y1 and P2Y12 antagonists
(
). The maximum aggregation extent in the absence of any antagonist
was taken as 100% and the remaining values were normalized to this
value. In the case of collagen, extent of aggregation in the presence
of SC57101, a fibrinogen receptor antagonist, was taken as zero.
Concentrations of agents used were: P2Y1 antagonists A3P5P, 1 mM (A) or
MRS2179, 100 µM (B,C); P2Y12 antagonists AR-C66096, 1 µM (A) or
AR-C69931MX, 100 nM (B,C). Each data point is the mean ± SE of 3 measurements with different donors.
