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Blood, 15 February 2004, Vol. 103, No. 4, pp. 1333-1341. Prepublished online as a Blood First Edition Paper on October 16, 2003; DOI 10.1182/blood-2003-03-0889.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human bloodFrom the Division of Cardiovascular Research, Department of Biochemistry, and the Division of Transfusion Medicine, Department of Haematology, University of Cambridge; the National Blood Service, Cambridge, United Kingdom; the Departments of Biochemistry and Human Biology, CARIM, University of Maastricht, Netherlands; and the Laboratory for Thrombosis Research, IRC, KU Leuven, Belgium.
The platelet glycoproteins (GPs) Ib, integrin  2 1, and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s–1), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb (12G1 Fab2) and 21 (6F1 mAb or GFOGER-GPP peptide). GPVI was found to be crucial for aggregate formation, Ca2+ signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting 21 revealed its involvement in regulating Ca2+ signaling, PS exposure, and aggregate size. Both GPIb and 21 contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb or 21. In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with 21 mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation.
The platelet response to exposed subendothelial matrix is fundamental to thrombosis and hemostasis. Uniquely, collagen, the most abundant vessel wall protein, mediates platelet adhesion and activation, localizing and regulating the hemostatic response at sites of injury. Discovering the molecular mechanisms that control platelet-collagen interaction is crucial for understanding the pathogenesis of arteriothrombotic diseases such as stroke and myocardial infarction. Under high shear rate conditions, the glycoprotein (GP) Ib/V/IX complex allows initial platelet rolling over von Willebrand factor (VWF) bound to subendothelial collagen fibers, and subsequently collagen receptors come into contact with their specific binding sequences in the collagen. For the next step, platelet arrest and activation, firm evidence exists of a role for only 2 receptors, integrin 21 and immunoglobulin superfamily member GPVI, despite the apparent redundancy in collagen receptors (for a review, see Siljander PR-M and Farndale RW1).
According to the 2-site, 2-step model, high-affinity interaction through
Recently, several lines of evidence strongly encouraged revision of the step-wise model, promoting the idea that GPVI is the primary collagen receptor. Integrin Following the deposition of platelets, their procoagulant response is essential for thrombus formation. Platelet aggregates are stabilized through fibrin formation, and newly generated thrombin recruits further platelets and activates other cells. At an advanced stage of activation, collagen-adherent platelets undergo a procoagulant transformation that includes the exposure of phosphatidylserine (PS), the secretion of coagulation factors, and the distinctive morphology of blebbing cells and microvesiculation.15 We have previously shown that GPVI is essential for triggering this response in collagen-adherent platelets, which is preceded by extensive intracellular Ca2+-signaling.16 Supporting this, prothrombinase activity was shown to correlate with GPVI density17 and genotype.18
This study was undertaken to clarify the role of the human collagen receptors GPVI and
Our results show that GPVI is crucial to collagen-induced Ca2+ responses, aggregate formation, and PS expression but that
Fibrillar type I collagen (Horm) from equine tendon was obtained from Nycomed (Munich, Germany). Apyrase (grade 7), heparin, and MRS2179, a P2Y1 antagonist, were from Sigma (St Louis, MO), H-Phe-Pro-Arg chloromethyl ketone (PPACK) was from Calbiochem (La Jolla, CA), and Fluo-3 and calcein acetoxymethyl esters were from Molecular Probes (Leiden, The Netherlands). Annexin V (Apoptest) labeled with Oregon Green 488 (OG488) was obtained from Nexins Research (Hoeven, The Netherlands). AR-C69931MX, an antagonist of the P2Y12 receptor, was from Astra-Zeneca (Charnwood, United Kingdom). Acetylsalicylic acid (ASA) was from Lorex Synthelabo (Maarssen, The Netherlands), and low-molecular–weight heparin (Fragmin) was from Pharmacia N.V. (Puurs, Belgium).
The GFOGER-GPP and CRP peptides were synthesized as described.20,23 ScFv antibodies 10B12 and 1C319 and mAb 12G1 against GPIb Blood collection
Blood from 9 healthy volunteers was collected in 40 µM PPACK in 0.1 vol saline, supplemented hourly with 10 µM PPACK. Donors had not taken medication for 2 weeks. Platelet counts were determined with a Coulter counter (Coulter Electronics, Hialeah, FL). Donors were genotyped for receptor polymorphisms: GPVI (a/b), Static platelet adhesion
Static platelet adhesion to CRP and fibrillar collagen was performed exactly as previously described,27 using cation-free conditions to examine the adhesive role of GPVI in the absence of Intracellular Ca2+ and thrombus volume measurements Acid citrate dextrose–platelet-rich plasma (ACD-PRP) was incubated with 7 µM Fluo-3 acetoxymethyl ester or calcein for 30 minutes at 37°C. Platelets were centrifuged with 0.1 vol ACD, washed once with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer containing 10 mM HEPES, 136 mM NaCl, 2.7 mM KCl, 2 mM MgCl2, 0.1% glucose, 0.1% bovine serum albumin (BSA), pH 6.6, centrifuged again with 0.1 vol ACD, and suspended in the original volume of PRP with HEPES buffer, pH 7.45. PPACK-anticoagulated blood was spiked with Fluo-3–labeled platelets at 10% of the original platelet count and with 30 µM PPACK. In control perfusions over collagen, 30% of 7 µM calcein-prelabeled autologous platelets were added. In real-time, Z-stacks of x-y scans were taken during the perfusion (box volume of 171.8 x 171.8 x 50 µm) at a 2-photon excitation wavelength of 800 nm and an emission of 485 to 515 nm (8% of full power) using a Radiance 2000 multiphoton laser scanning fluorescence microscope system (BioRad, Hemel Hempstead, United Kingdom), equipped with a pulsed sub-picosecond Tsunami Ti:sapphire laser (Spectra-Physics, Mountain View, CA). Two-photon images were analyzed with Laserpix software (Bio-Rad). Flow experiments and image recording Whole blood perfusion experiments were performed essentially as described for mouse blood.28 Briefly, glass coverslips were coated with collagen fibers (12.5 µg/cm2) and blocked with HEPES buffer, pH 7.45, containing 1% BSA and 1% glucose. The blood was placed in a syringe and perfused over the coverslip through a transparent 50 µm deep chamber using a pulse-free pump,29 at a shear rate of 1000 s–1 for 4 minutes. Blood was incubated for 15 minutes before perfusion with various concentrations of scFv 10B12 or 1C3, 10 to 20 µg/mL 6F1, 500 µg/mL GFOGER-GPP, 10 µg/mL 4B4, 20 µg/mL Fab2 fragment 12G1, ASA (100 µM), MRS2179 (40 µM), and AR-C69931MX (20 µM) or 1 U/mL apyrase, as described. Where indicated, autologous Fluo-3–labeled platelets were added to the blood before antagonists. Microscopic phase-contrast and fluorescent images from Fluo-3–labeled platelets were recorded in real-time using a Visitech digital imaging system (Sunderland, United Kingdom) equipped with 2 intensified, charge-coupled device (CCD) cameras.30 After perfusion, flow chambers were rinsed at the same flow rate for 4 minutes with HEPES buffer, pH 7.45, supplemented with 1 U/mL heparin and 2 mM CaCl2. Phase-contrast and fluorescent images were collected with a 40x UV-transparent objective and 15x to 20x image magnification. Exposure of PS was detected after incubation of the slide with 100 µL HEPES/CaCl2 buffer, pH 7.45, containing OG488-labeled annexin V (1 µg/mL) for 5 minutes. Antibodies and antagonists were also added to the rinsing buffer and the annexin V incubation. Image analysis Surface coverage from phase-contrast images was analyzed using Image-Pro (Silver Spring, MD) software version 4.1, and it was analyzed from platelets stained with OG488-annexin V using Quanticell software (Visitech). Distribution of aggregate sizes in phase-contrast images was measured using Leica QWin image analysis software (Leica Imaging Systems, Cambridge, United Kingdom). Changes in Fluo-3 fluorescence from individual platelets were converted to [Ca2+]i as described.31 To provide a measure of the proportion of procoagulant cells independent of platelet deposition, the ratio of annexin V–binding surface coverage to phase-contrast surface coverage was calculated and was termed procoagulant index (Pi). Although the procoagulant area was overestimated through fluorescent glare in the optics, Pi provided a means of distinguishing the effects of treatments on procoagulant expression from those on platelet deposition. Experimental design and statistics Each experimental condition was tested on at least 3 occasions using blood from different donors, and each donor provided blood for at least 10 perfusion experiments. Control conditions were included for each blood sample, together with other permutations of conditions. For each perfusion surface, images from 9 random microscopic fields were collected, and the average percentage area covered by adherent platelets was measured either by phase-contrast or by OG-labeled annexin V fluorescence. Phase-contrast, fluorescent surface coverage (mean ± SE) and Pi were compared among all experimental groups using analysis of variance (ANOVA), with Newman-Keuls or Dunnett posttesting. Data derived from the same donors were compared by paired sample t tests or ANOVA. The effect of ADP blockade under different conditions was tested by 2-way ANOVA.
Image analysis provided estimates (area and roundness of separate features, such as platelets and aggregates) of thrombus size and shape. Features were segmented digitally and measured with minimal operator intervention. From an average of 9 images, using 3 different donors, at least 200 different features were measured for each treatment, the effects of which were determined by
Blocking of GPVI abolishes aggregate formation, Ca2+ signaling, and PS exposure without eradicating primary platelet adhesion
PPACK-anticoagulated whole blood was perfused at high shear rate (1000 s–1) over a surface covered with native collagen type I fibrils. Phase-contrast and fluorescence microscopic images were captured to monitor 2 independent parameters of thrombus formation measured as the surface coverage of deposited platelets and of procoagulant, PS-exposing platelets that stained with OG488-conjugated annexin V. In control perfusions, platelets rapidly adhered to the collagen fibers, forming aggregates that later coalesced (Figure 1A). The deposition of platelets measured by surface coverage was linear in time, indicating regular and continuous scavenging of platelets (Figure 2A). To confirm that the measuring surface area represents a linear progression of thrombus build-up, the thrombus volume was measured by using calcein-labeled platelets from the same donor and real-time scanning of the 2-photon fluorescence signal, which even at more than 50 µm penetration was typically not distorted by the flowing blood. Again, thrombus volume increased linearly in time (Figure 2B). No differences in platelet deposition were observed within the donor population, relating to genetic polymorphisms of GPVI,
Blocking GPVI by 10B12 inhibited CRP- and collagen-induced platelet aggregation in vitro.19 Static platelet adhesion to CRP was abolished at 5 µg/mL and decreased by 80% ± 4% (n = 4) to collagen fibers at 25 µg/mL. However, under flow, a concentration curve for 10B12 (Figure 4) showed that, up to 300 µg/mL, surface coverage with platelets did not significantly decrease (r2 = 0.22; P = .42), though, dramatically, platelet aggregates were absent (Figure 1B-D). Biosensor data indicated that at 300 µg/mL 10B12, occupancy of recombinant GPVI had reached more than 97% (data not shown). The predominance of single platelets with increasing 10B12 concentrations, obvious to the eye, was quantified by image analysis as almost complete elimination of larger aggregates (P < .0001) at 50 µg/mL 10B12, and the mean area was reduced to 37 ± 2 µm2 from 176 ± 16 µm2 (P < .001) (Figure 3B). In contrast to platelet adhesion, surface coverage of PS-exposing platelets decreased progressively with the 10B12 concentration with an IC50 value of 23 µg/mL (r2 = 0.94; P < .01) (Figure 4), and blebbing platelets were no longer visible. At the highest concentration of 10B12 tested, the Pi was reduced to 0.02 ± 0.005 (P < .001), indicating strong blockade of GPVI function.
ScFvs 1C3 and 10B12 recognizes distinct epitopes, and 1C3 is incapable of blocking static platelet adhesion to CRP or collagen-induced platelet aggregation.19 As expected, 1C3 (100 µg/mL) did not alter either the surface coverage (Figure 1E) or the size and morphology (Figure 3C) of the formed thrombi. However, 1C3 halved the PS expressing surface coverage (Figure 4), suggesting that the 1C3-binding site is relevant for GPVI function, possibly by influencing receptor clustering or dimerization. The Pi obtained with 1C3 was reduced to 0.24 ± 0.07 (P < .05, relative to control), a significantly higher Pi than with 100 µg/mL 10B12 (P < .001). Coinhibition with 10B12 and 1C3 did not change surface coverage from that of 10B12 alone (data not shown). To study the dynamics of platelet activation, Ca2+ signal generation was measured in real-time under flow from single, Fluo-3-labeled platelets coming in contact with collagen. Control platelets exhibited a strong Ca2+ response, sometimes preceded by an initial Ca2+ spike (Figure 5A). A submaximal dose (50 µg/mL) of 10B12 decreased the Ca2+ signal amplitude (Figure 5B). Although blocking GPVI did not abolish primary adhesion, the increased translocation of platelets across the collagen surface was observed during Ca2+ measurements. Increasing the 10B12 concentration to 300 µg/mL reduced the Ca2+ signal slightly further (Figure 5C). No difference from controls was observed in the averaged Ca2+ response with 1C3 (100 µg/mL), though Ca2+-spiking occurred in individual platelets (Figure 5D). Together, these data indicate that blocking of GPVI with 10B12 potently suppressed collagen-dependent platelet activation pathways without eradicating primary adhesion.
Blocking of GPIb or
High levels of VWF and high
The importance of GPIb in primary adhesion4 was reconfirmed under the shear rate conditions used here. Using 12G1 Fab2 at a maximally effective dose of 40 µg/mL, the overall surface coverage was reduced by 50% ± 14% (Figure 6), which confirms that at (arterial) shear rates of approximately 1000 s–1, platelet adhesion to collagen is only partially dependent on VWF-GPIb interaction.38,39 Although in 2 donors surface coverage was strongly inhibited (63% and 67%), 2 gave a weaker reduction in thrombus formation (38% and 45%), but the differential response did not correlate with –5C/T polymorphism. For all donors, blocking of GPIb strongly influenced platelet tethering. GPIb
Inhibition of
Inhibition of
Combined inhibition of GPVI with either GPIb or
Coinhibition of GPVI with 10B12 (100 µg/mL) and of GPIb with 12G1 Fab2 (40 µg/mL) resulted in almost complete abolition of platelet adhesion, with remaining single platelets covering not more than 2% of the surface; PS-exposing platelets were hardly detected (Figure 7A-B). Similarly, coinhibition of GPVI and
In contrast, combined blocking of GPIb and
Blocking of ADP receptors further inhibits thrombus formation restricted by GPVI or ADP regulation of thrombus size has been attributed to the platelet P2Y12 purinergic receptor; P2Y1 regulates the onset of thrombus formation.41 To obliterate any effects of ADP, we used apyrase in conjunction with specific P2Y12 and P2Y1 inhibitors. Full inhibition of ADP- or TxA2-mediated (inhibited by ASA) effects reduced the surface coverage by 43% (P < .001). The morphology of platelet deposition was similar to that under GPVI blockade: a layer of single platelets remained on the collagen (Figure 1H), and aggregates were eradicated (Figure 3E). However, combined blockade of ADP and TxA2 actions had no effect on Pi (0.53 ± 0.15 [not significant]) or platelet blebbing, though it slightly reduced the Ca2+ response (Figure 5G). Inhibiting the ADP pathway further decreased surface coverage when combined with GPVI blockade. Irrespective of the 10B12 concentration (50 or 300 µg/mL), ADP receptor antagonism caused an additional reduction in platelet surface coverage (55%; P < .001) (Figure 8A), suggesting that the presence of ADP was not solely derived from GPVI-dependent secretion. Because GPVI inhibition itself abolished aggregate formation, the additional blockade of ADP receptors had no further effect on the mean feature size: 24 ± 2.6 µm2 (ADP + GPVI blockage) and 23 ± 1.8 µm2 (GPVI blockade alone) (Figure 8B). When applied together with 10B12, ADP receptor blockade reduced the number of PS-exposing platelets by attenuating platelet deposition because the Pi did not significantly decrease.
When the ADP receptors were coinhibited with
The present study shows that, in humans, collagen-induced thrombus formation under high shear rate requires the orchestrated interplay of several platelet receptors. Using antibodies and peptides allowed clarification of the complementary and overlapping roles of the GPIb/V/IX complex, 21 integrin, GPVI, and the platelet purinergic receptors in the processes of platelet adhesion, aggregation, and procoagulant activity. Our results, though formally in line with the original 2-step adhesion-activation model, also revealed its oversimplification. Although the main role of GPIb is to provide initial contact with collagen-bound VWF, it stabilizes the anchorage of thrombi on collagen together with 21. GPVI is involved in GPIb- and 21-mediated adhesion through activation, and it is the main signaling receptor during thrombus formation. Conversely, 21 assists GPVI in signaling. Complete abrogation of adhesion and subsequent activation thus requires simultaneous inhibition of GPVI together with either GPIb or with 21.
We find that GPVI has a crucial role in regulating human thrombus growth. Inhibiting GPVI resulted in the complete loss of platelet aggregates and the full inhibition of collagen-induced Ca2+ mobilization and PS exposure. In contrast, the adhesion of single, nonaggregated platelets was not significantly diminished unless ADP was also antagonized, probably eliminating the contribution of
Both GPIb and
The present model of human thrombus formation deviates in certain respects from that derived from murine data. Although murine thrombus formation seems to rely more on GPVI, it was recently shown that the interplay of After the primary platelet-collagen contact, platelets form aggregates or become procoagulant; both processes contribute to full-blown thrombus formation. We defined 2 conditions in which the formation of thrombi was inhibited: blocking GPVI and blocking ADP- and TxA2-mediated events. Inhibiting GPVI suppressed aggregate formation and signaling processes, detected as the greatly reduced Ca2+ and procoagulant response. However, ADP/TxA2 antagonism reduced Ca2+ signaling only slightly, though no aggregates were formed, with no effect on Pi. GPVI regulates aggregate formation through autocrine ADP/TxA2 secretion, in part through synergism with Gi/q-coupled receptors.53 We found that ADP contributed to aggregation and primary adhesion but that its influence was not eliminated by GPVI inhibition. This implies that ADP was derived from sources other than GPVI-stimulated platelets.
Platelet aggregation was mostly accompanied by high Ca2+ signaling and PS exposure, even when blocking shear-dependent adhesion through GPIb. The platelets that escaped inhibition were activated, as demonstrated by an unchanged Pi. A notable exception occurred with the blocking of | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
(–/–) mice, generating a deficiency in the coexpressed GPVI, completely lacked a response to collagen under flow, as did wild-type mouse blood incubated with an anti-GPVI monoclonal antibody (mAb), indicating a key role for GPVI.
2 analysis. Data are plotted as frequency distributions with mean values per field (± SE). Prism (GraphPad Software, San Diego, CA) was used throughout for multiple comparisons and linear regression. 
), 2 to 8 (
), 8 to 40
(
), 40 to 130 (
), and more than 130 platelets (
). (A) Control. (B) 10B12 at 50 µg/mL. (C) 1C3 at 100 µg/mL. (D) ADP receptor and TxA2 blockade. (E) 6F1 at 20 µg/mL. (F) GFOGER-GPP at 500 µg/mL. Data (A-F) are mean ± SE from 3 donors. Statistical significance compared with the control was established from contingency tables (
