Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:3033-3043

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Siljander, P.
Right arrow Articles by Lassila, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Siljander, P.
Right arrow Articles by Lassila, R.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CALCIUM COMPOUNDS
*CALCIUM, ELEMENTAL
*L-TYROSINE
*MAGNESIUM COMPOUNDS
*MAGNESIUM, ELEMENTAL
*TRISODIUM CITRATE
Related Collections
Right arrow Biochemistry and metabolism
Right arrow Platelets
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:3033.)
© 1999 American Heart Association, Inc.

Thrombosis

Studies of Adhesion-Dependent Platelet Activation

Distinct Roles for Different Participating Receptors Can Be Dissociated by Proteolysis of Collagen

Pia Siljander; Riitta Lassila

From the Wihuri Research Institute (P.S., R.L.) and the Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki (P.S.), Helsinki, Finland.

Correspondence to Dr Riitta Lassila, Wihuri Research Institute, Kalliolinnantie 4, SF-00140 Helsinki 14, Finland. E-mail riitta.lassila{at}wri.fimnet.fi


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—The molecular differences between native-type collagen type I fibrils (NC) and their pepsinated monomers (PC) were used to uncover receptors involved in platelet-collagen interaction along the adhesion-activation axis. The platelet-depositing capacity of NC and PC under blood flow and their adhesive properties and respective morphologies, aggregation, procoagulant capacity, and tyrosine phosphorylation were compared under different cationic milieus, including or excluding the glycoprotein (GP) Ia/IIa. NC was consistently a more preferable and activating substrate than PC during flow (5 minutes) and in platelet aggregation. In PPACK-treated blood, both NC (3.3-fold) and PC (2.7-fold) increased platelet attachment on elevation of the shear rate from 500 to 1640 s-1, whereas in citrated blood, adhesion and thrombus growth on PC were negligible under the high shear rate, unlike on NC (1.9-fold increase). The complete lack of platelet deposition on PC in citrated blood could be overcome by restoring physiological Mg2+ concentration, and in contrast to NC, platelets interacting with PC were highly dependent on Mg2+ during adhesion, aggregation, and procoagulant response. Monoclonal antibody (mAb 131.7) against GP IV inhibited platelet deposition to NC in citrated blood (2 minutes) by 49%, which was not further increased by coincubation with mAb against GP Ia (6F1). These results stress the importance of GP Ia/IIa in shear-resistant platelet deposition on collagen monomers. In native fibers, however, the preserved quaternary structure with telopeptides activates additional platelet receptors capable of substituting GP Ia/IIa and GP IV.


Key Words: hemostasis • platelet • collagen • receptor • divalent cations


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
On exposure of subendothelial collagen to flowing blood, adhering platelets, with their subsequent activation and procoagulant capacity, initiate hemostasis and thrombosis.1 2 3 4 Pathophysiological platelet-collagen interactions include bleeding disorders due to either defective receptors or autoantibodies2 3 5 or to impaired collagen synthesis,6 7 8 indicating several functional determinants on both the platelets and collagen.

Thus far, glycoprotein (GP) Ia/IIa, integrin {alpha}2ß1, has been recognized as the major platelet receptor for collagen in primary adhesion, and accordingly, dysfunctional GP Ia/IIa associates with prolonged bleeding tendencies in patients.9 10 11 Although GP IV has been implicated in the very early phases of adhesion, the impact of GP IV,12 13 14 as well as the other receptor candidates,15 16 17 on hemostasis has remained unsettled. GP VI is involved in collagen-induced platelet activation, evidenced both by a poor aggregation response in patients lacking this receptor5 and by collagen-related peptides that likely induce strong platelet aggregation in normal platelets via this receptor.18 19 However, patients lacking GP VI have only mild bleeding tendencies,5 and under blood flow, the collagen-related peptides are unable to retain firm adhesion.20 Qualitative differences in collagen preparations complicate the identification of the determinants in platelet-collagen interaction. The term "native collagen fibers" has been used to cover a spectrum of preparations, from suspensions of native fibrils (eg, Horm) to dialysed fibrils consisting of salt-soluble tropocollagens, but also "fibrils" formed after dialysis of protease-extracted collagen monomers. However, the removal of telopeptides, eg, by pepsin, prevents the formation of fibrils, resulting instead in small nonbanded fibrous aggregates,21 and the severity of the impaired fibril formation depends on the extent of the pepsin digestion.22 Thus, the structural differences in collagen preparations are at least partly responsible for the inconclusive data on the determinants of the platelet-collagen interaction. Moreover, studies focusing on these effects under blood-flow conditions have been limited,23 most of the studies having used static adhesion and traditional platelet aggregation techniques.24 25

To obtain more information about the platelet-recruiting capacity and activating potential of type I collagen, the collagen monomers derived from fibrils by extensive pepsin treatment (eliminating telopeptides and quaternary structure) were used as a tool to investigate the responsible determinants. These fibrils and monomers were compared as substrates in experiments that dissociated the hemostatic events evolving from the platelet-collagen interaction under flowing blood: adhesion, aggregate formation, tyrosine phosphorylation, and procoagulant activity.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
5-Hydroxy(G-3H)tryptamine creatinine sulfate (5-HT) was obtained from Amersham International plc. Aurin tricarboxylic acid (ATA, MW 473.4) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) peptide were from Sigma. Horseradish peroxidase (HRP)–conjugated mouse anti-human von Willebrand factor (vWF), HRP-conjugated goat anti-mouse IgG antibodies, and monoclonal antibody (mAb) AN51 (against GP Ib) were from Dakopatts A/S; mAbs Gi9 (against GP Ia), SZ2 (against GP Ib), and SZ22 (against GP IIb) were from Immunotech SA; and 4G10 (against phosphotyrosine) was from Upstate Biotechnology, Inc. S-2238 was from Chromogenix AB and Thromborel S from Behringwerke AG. Bovine {alpha}-thrombin was from Dade (Baxter Healthcare Co). mAbs 131.7 (against GP IV) and 6F1 (against GP Ia, {alpha}2) were kind gifts from Dr Narendra Tandon (Otsuka America Pharmaceutical, Inc, Rockville, Md) and Dr Barry Coller (Mount Sinai Medical Center, New York, NY), respectively, and vWF was obtained from Dr Thierry Burnouf (Center Regional de Transfusion Sanguine, Lille, France).

Preparation of Collagen
Collagen type I fibrils from bovine Achilles’ tendon were extracted with 0.5 mol/L acetic acid and salt-precipitated with 1.7 mol/L NaCl, essentially as previously described.26 To obtain collagen monomers (PC) devoid of quaternary structure and telopeptides, native-type collagen fibrils (NC) were extensively treated with pepsin [3 additions of pepsin (Sigma Chemical Co), 1 mg/mL, rotation for 3 weeks at 4°C] and salt-precipitated. After the collagen stock solutions were centrifuged at 100 000g for 1 hour at 4°C, only 2% of NC (acid-insoluble collagen) but >90% of PC (acid-soluble collagen) remained in the supernatant. The collagen concentrations were determined by a modified hydroxyproline assay27 or with Sircoll dye from Biocolor Ltd. The structural properties of the collagen preparations were analyzed by transmission electron microscopy (TEM) (JEOL 1200EX). Collagen stocks were immobilized onto gold grids supported with carbon-coated polyvinyl formal plastic film, and negatively stained with 1% potassium phosphotungstic acid, pH 7.3, for 1 minute at 22°C.

Blood Collection
The study was approved by an institutional review board. Blood was obtained from healthy volunteers who had not used any medication during the previous 10 days. Nine volumes of free-flowing blood were collected into 1 volume of either (1) 30 µmol/L (final concentration) D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) (Calbiochem-Novabiochem Corp); (2) 90 mmol/L sodium citrate, additionally adjusted depending on the donor’s hematocrit28 ; or (3) acidic citrated dextrose, pH 4.5, for gel-filtered platelets (GFPs). The anticoagulants were chosen to yield various concentrations of divalent cations that affect the function of GP Ia/IIa.10 29 The concentration of ionized magnesium in blood anticoagulated with citrate was 8 µmol/L, and with PPACK, it was 0.62 mmol/L, as measured with a Microlyte 6 electrolyte analyzer (Kone Instruments Corp). The concentrations of ionized magnesium in platelet-rich plasma (PRP) were 4 µmol/L in citrate and 0.61 mmol/L in PPACK. The concentrations of ionized calcium were 86 µmol/L in citrated and 1.37 mmol/L in PPACK-treated blood, and 79 µmol/L and 1.37 mmol/L in PRP, respectively. Platelet counts and hematocrit were determined by a Thrombocounter Coulter T-540 (Coulter Electronics, Inc).

Platelet Preparation
GFPs for adhesion studies were prepared from PRP after a single washing step in the presence of prostaglandin E1 (25 ng/mL) and apyrase (1 U/mL) (Sigma) by elution of the platelet suspension through a Sepharose 2B column (Pharmacia LKB Biotechnology Inc) in HEPES buffer without divalent cations.30 Platelets were labeled with [3H]5-HT (specific activity, 13 Ci/mmol), 12 nmol/mL of PRP, or GFPs for 15 minutes at 37°C. The [3H]5-HT labeling method has been validated previously.31

Coverslips With Immobilized Collagen
Thermanox coverslips (NUNC), to be used during the same day, were coated by spraying 10 times with NC or PC (0.36 mg/mL) alternating with neutralizing buffer and then incubated (90 minutes, 35°C) to induce in situ fibril formation.32 For observation under light microscope, the coverslips were stained with Coomassie brilliant blue R250.

Platelet Deposition on Immobilized Collagen Under Flow Conditions in Whole Blood
To study platelet deposition (adhesion-dependent platelet aggregate formation), whole blood was perfused in parallel-plate perfusion chambers with defined rheological characteristics.31 33 34 After the PRP was labeled, the blood was reconstituted without adjustment of the platelet counts. When indicated, the PRP was incubated with mAb 6F135 (final concentration, 10 µg/mL) and/or mAb 131.736 (5 µg/mL) for 20 minutes at 22°C before the blood was reconstituted and then allowed to stabilize for 30 minutes. The prewarmed (37°C) blood aliquots were recirculated over coverslips for 5 minutes at shear rates of 500 or 1640 s-1. Then the samples were briefly perfused and rinsed with PBS, and the deposited 3H activity was measured in a liquid scintillation counter (1414 Rackbeta, Wallac), as described previously.31 The perfusions were completed within 3.5 hours of blood collection.

Scanning electron microscopy (SEM; JEOL SEM-820) was used to analyze the morphology of platelet deposition and adhesion. The samples were fixed with 2.5% phosphate-buffered glutaraldehyde at 22°C for 2 hours. After rinsing with PBS, the samples were dehydrated, critical point–dried under CO2, and sputter-coated with gold or platinum.

Platelet Adhesion on Immobilized Collagen
GFPs (100x106/mL) were labeled as described. Collagen-coated coverslips were placed in 24-well flat-bottom multidishes (NUNC) precoated with 2% human serum albumin, and GFPs (0.5 mL) were incubated stationary for 30 minutes at 22°C in the presence of 2 mmol/L of either Ca2+ or Mg2+.10 Afterwards, the coverslips were rinsed in PBS and subjected to scintillation counting. This platelet density and stationary conditions were chosen to promote adhesion without inducing platelet-platelet interaction. When indicated, GFPs were preincubated for 30 minutes35 with mAb Gi9 (20 µg/mL) or 6F1 (10 µg/mL), which had been dialyzed to remove NaN3.

Platelet Aggregation
Aggregation of platelets (300x106 mL in suspension or in PRP anticoagulated with either acidic citrated dextrose, pH 6.5, or PPACK) was measured turbidimetrically in a dual-channel aggregometer (Payton Associates Inc). The samples were stirred at 700 rpm and preincubated for 1 minute at 37°C before the addition of collagen. The rates of primary aggregation (min-1) and maximal aggregation (%) at 5 minutes were assessed.

Tyrosine Phosphorylation
GFP suspensions (500x106/mL), preincubated with 300 µmol/L of KQAGDV, were stimulated in an aggregometer with 1.5 µg/mL of NC or preformed PC "fibrils" (HEPES buffer, 1.5 hours, 35°C) in the presence of 2 mmol/L of either Ca2+ or Mg2+. At selected time points, an equal volume of 2x SDS Laemmli sample buffer with 3 mmol/L sodium orthovanadate was added. The samples (20 µL) were electrophoresed on a 7.5% SDS-polyacrylamide gel and transferred onto nitrocellulose membrane (Probind 45, Pharmacia). After blocking, the membranes were incubated with 0.2 µg/mL of 4G10 and HRP-conjugated goat anti-mouse antibody. The tyrosine phosphorylation patterns were visualized with the enhanced chemiluminescence technique (ECL kit, Amersham). The blots were stripped and then reprobed with SZ22 to control the platelet amounts on different lanes.

Assessment of Procoagulant Activity of Collagen-Adherent GFPs
After adhesion of GFPs on either NC or PC in the presence of 2 mmol/L of either Ca2+ or Mg2+, the coverslips were washed and incubated for 10 minutes with PBS containing 0.9 mmol/L CaCl2. Then a 1:20 dilution of platelet-poor plasma (PPP) was added, and the generation of thrombin was enhanced by trace amounts of tissue thromboplastin (1:5000 final dilution of Thromborel S) under agitation for 10 minutes at 37°C.37 From each well, 180 µL of supernatant was reacted with a chromogenic substrate, S-2238 (200 µmol/L), for 6 minutes. Absorbance was measured at 405 nm (Labsystems Multiscan MCC). A control was included by incubating PPP and thromboplastin over collagen-coated coverslips without adhered platelets. This control value was deducted from the values obtained with platelet-adhered coverslips. The thrombin amounts formed were calculated by use of known concentrations of bovine {alpha}-thrombin (Dade) in a standard curve.

Statistics
The data are presented as mean±SD and were analyzed with a paired Student’s t test. Regression analysis was applied to study the association between the adhesion results. All assays were done in duplicate, and n refers to the number of donors. Because of the great interindividual differences in platelet responses to collagen,38 all comparisons between NC and PC in a given experiment were performed in samples from the same donor.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Collagen Structure
The Coomassie blue–stained collagen-coated coverslips were viewed by light microscopy after the treatment to induce fibrils. These were thick bundles of collagen fibrils that clearly differed from segmented strands of collagen monomers (Figure 1Down, A and B). The collagen preparations were negatively stained and analyzed for periodicity in TEM to verify that the native type I collagen was in the form of true fibrils. Indeed, this preparation displayed periodic fibrils, whereas the PC monomers failed to do so (Figure 1Down, C and D), even after the treatment to induce "fibrils."



View larger version (190K):
[in this window]
[in a new window]
  		Figure 1. Light micrographs of collagen-coated coverslips (A and B) and TEM micrographs of negatively stained collagen preparations (C and D), illustrating the differences in collagen structure. The native-type collagen I fibrils (A and C) expressed periodic structure (C), which was lacking in monomers (B and D). Despite the fibril-forming treatment, the monolayers remained as nonbanded fibrous aggregates (D). Light microscopy magnification x20, bar=10 µm; TEM magnification x40 000, bar=200 nm.

Whole-Blood Perfusion Studies
To dissociate the involvement of different receptors during platelet-collagen interaction, the platelet-recruiting capacity of NC and PC was compared under various shear rates and anticoagulants: a low to intermediate shear rate of 500 s-1 and a high shear rate of 1640 s-1 and blood anticoagulated with either citrate (86 µmol/L Ca2+, 8 µmol/L Mg2+) or 30 µmol/L PPACK (normal cations; 1.37 mmol/L Ca2+, 0.62 mmol/L Mg2+), respectively. Under the perfusion conditions tested, platelets predominantly preferred NC over PC (Figure 2ADown).



View larger version (81K):
[in this window]
[in a new window]
  		Figure 2. A, Platelet deposition (mean±SD) on NC and PC during whole-blood perfusions for 5 minutes under conditions favoring platelet-platelet interaction and activation (shear force, 37°C). More platelets deposited on NC (hatched bars) than on PC (solid bars) regardless of the anticoagulant (PPACK, n=6, or citrate, n=4) or the shear rate (500 or 1640 s-1) (*P<0.05; **P<0.0001). LS indicates low shear rate (500 s-1); HS, high shear rate (1640 s-1). B and C, SEMs of the perfusion channel on PC with platelet deposition at either the low (500 s-1) (B) or the high (1640 s-1) (C) shear rate from blood perfusions anticoagulated with citrate. Magnification x105; bar=100 µm.

In PPACK-anticoagulated blood, platelet deposition was not significantly larger on NC than on PC at the low shear rate of 500 s-1 (5.4±2.7 versus 3.7±1.7x106/cm2), but at the high shear rate of 1640 s-1, almost 2 times more platelets deposited on NC than on PC (17.8±10.3 versus 10.0±6.8x106/cm2) (Figure 2AUp). The shear-dependent increase in platelet deposition was of similar magnitude on both substrates: 3.3-fold on NC and 2.7-fold on PC, suggesting that the vWF-mediated platelet deposition was not markedly affected by the differences between NC and PC. This result was further confirmed by use of ATA as an inhibitor for vWF-mediated platelet deposition on collagen.39 At the high shear rate, ATA inhibited platelet deposition in a similar manner for both PC and NC, by {approx}40% (data not shown).

In citrated blood devoid of Mg2+, which is important for GP Ia/IIa activity, the total platelet deposition diminished on both substrates (Figure 2AUp), and the platelet-recruiting differences between NC and PC were accentuated. Over 3-fold more platelets deposited on NC than on PC at the shear rate of 500 s-1 (3.9±0.7 versus 1.2±0.4x106/cm2). The difference between the collagens was even more profound when the shear rate was elevated to 1640 s-1, which resulted in 15 times larger platelet deposition on NC than on PC (7.6±3.1 versus 0.5±0.2x106/cm2). NC showed a shear-induced increase in platelet deposition, in contrast to PC, on which platelet adhesion and subsequent aggregation were severely impaired at the high shear rate. This effect was also clear in the SEMs of the perfusion channels subjected to the low versus high shear rate: whereas full aggregates were able to form on PC under the low shear force (Figure 2BUp), platelets and platelet aggregates were absent under the high-shear-rate conditions (Figure 2CUp).

Because of the lack of platelet deposition on PC under the high shear rate, we assessed the binding of vWF from citrated plasma to NC and PC under both stationary conditions and flow. Detected with HRP-conjugated mAb, NC bound more vWF than PC under stationary conditions. However, in flow under high-shear-rate conditions, vWF binding from citrated PPP with washed and glutaraldehyde-fixed red blood cells28 did not differ (data not shown). Furthermore, incubation of the PC surface with vWF (10 µg/mL) before the perfusion did not improve the defective platelet deposition (data not shown). However, when the physiological level of Mg2+ cations was restored in citrated blood (by addition of 7 mmol/L MgCl2, Microlyte 6 analyzer), platelet deposition on PC was corrected, reaching up to 84% (66±20%, n=4) of that displayed in PPACK-treated blood under the high-shear-rate conditions. The restoration of physiological magnesium concentration allowed platelet deposition on PC to reach 61% and 83% of the NC-induced platelet deposition at the high and the low shear rates, respectively (n=2). In PPACK blood at 3 minutes of perfusion, 6F1 dropped the deposition to PC by 92% (n=1). Interestingly, additional determinants of NC could circumvent the functionally restricted GP Ia/IIa, as evidenced by the moderate platelet deposition in citrated blood at both shear rates. Furthermore, at the low shear rate, the platelet deposition on NC in citrated blood did not essentially differ from platelet deposition in PPACK blood (P=0.16). Finally, the inhibitory effects of mAbs against GP IV and GP Ia were tested on platelet deposition to NC after 2 minutes of perfusion. mAb 131.7 inhibited 49±16% (n=5) of platelet deposition on NC at high shear rate in citrated blood. Under these conditions, the addition of 6F1 did not further inhibit platelet deposition (data not shown).

SEM supported the results of [3H]5-HT–labeled perfusions, illustrating both the surface coverage and the morphology of deposited platelets (Figure 3Down). At the low shear rate, irrespective of the collagen surface, and in the same anticoagulant, platelets and aggregates were fairly evenly distributed, but the size of the aggregates varied (Figure 3Down, A through D). The high shear rate increased the morphological differences, which depended on both the anticoagulant and the substrate (Figure 3Down, E and F). The relatively low PPACK concentration was chosen to allow adhesion-dependent activation events and possible formation of local fibrin. In PPACK-anticoagulated blood, platelet deposition on NC ranged from single platelets to multilayered aggregates (Figure 3EDown), whereas platelet deposition on PC seemed more uniform (Figure 3FDown). Strand-like structures, most likely representing locally formed fibrin, were found between the aggregates. However, it has been shown previously that at this PPACK concentration, circulating thrombin–antithrombin III complexes were not elevated after the perfusion.31 In citrated blood, platelet recruitment on NC resembled that on PC in PPACK blood (Figure 3GDown). Contrasting the evenly covered perfusion channel with PC at 500 s-1 (Figure 3Down, B and D) or with NC under all conditions (Figure 3Down, A, C, E, and G), platelets were virtually absent on PC in citrated blood under the high shear rate (Figure 3HDown). Figure 3HDown shows the few platelets deposited only close to the channel edges (Figure 2CUp), where the shear rate decreases.



View larger version (131K):
[in this window]
[in a new window]
  		Figure 3. SEMs illustrating the differences in coverage and morphology of platelet depositions after blood perfusions over NC and PC under various anticoagulants and shear rates: A, NC, 30 µmol/L PPACK, 500 s-1; B, PC, 30 µmol/L PPACK, 500 s-1; C, NC, citrate, 500 s-1; D, PC, citrate, 500 s-1; E, NC, 30 µmol/L PPACK, 1640 s-1; F, PC, 30 µmol/L PPACK, 1640 s-1; G, NC, citrate, 1640 s-1; and H, PC, citrate, 1640 s-1. Magnification x1050; bar=10 µm. Note the very few platelets in H located at the channel edge.

Altogether, the necessity of functional GP Ia/IIa was obvious in platelets adhering to PC under high-shear-rate conditions: The addition of Mg2+ to blood was able to correct the lacking platelet deposition in citrated blood, and a mAb against GP Ia significantly inhibited platelet deposition in PPACK blood. The structural determinants of NC, however, were able to attract platelets via receptors other than GP Ia/IIa and GP IV and to enhance platelet activation.

Adhesion Studies
Because platelet deposition depended on the shear-resistant buildup of the thrombus on adhesion, we next studied the primary adhesion of platelets on PC and NC in the presence of 2 mmol/L Mg2+ or Ca2+ and specifically the role of GP Ia/IIa therein.10 In the presence of Mg2+, PC was consistently a significantly preferable substrate for platelet adhesion than NC (Figure 4Down). The adhesion to PC correlated well with the adhesion to NC (r=0.83, P<0.005), indicating the involvement of the same platelet receptor, ie, GP Ia/IIa, in adhesion to both PC and NC. On the contrary, switching the cations from Mg2+ to Ca2+ strongly decreased platelet adhesion to PC, resulting in 2.4 times larger adhesion to NC than to PC, probably because of the closure of GP Ia/IIa for collagen.40 In sharp contrast to PC, switching the cations from Mg2+ to Ca2+ enhanced platelet adhesion on NC (Figure 4Down). Ca2+-dependent platelet adhesion to NC did not correlate with Mg2+-dependent adhesion to PC (r=0.41), but it did correlate with the adhesion to NC in the presence of Mg2+ (r=0.76, P<0.01), suggesting distinct receptors for NC under the differing divalent cation milieus as well as an interplay between GP Ia/IIa and another receptor(s).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 4. Adhesion of GFPs (100x106/mL, 500 µL) on NC (hatched bars) and PC (solid bars) after 30 minutes, at 22°C, stationary and in the presence of 2 mmol/L of either Mg2+ or Ca2+. Data represent mean±SD; n=15; *P<0.05; **P<0.0001.

Blocking GP Ia with mAbs (6F1 or Gi9, both IgG1 subclasses) decreased Mg2+-dependent platelet adhesion to PC (63% and 52%, respectively) (Figure 5ADown). The extent of this inhibition was similar to that caused by the switching of Mg2+ to Ca2+, and in the presence of Ca2+, 6F1 did not further inhibit adhesion to PC. The mAbs did not inhibit platelet adhesion on NC when either Mg2+ or Ca2+ was provided (Figure 5BDown). In contrast, in the presence of Mg2+, 6F1, but not Gi9, rather increased adhesion on NC. As a control, mAbs against GP Ib did not affect adhesion on either PC or NC (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Effects of mAbs against GP Ia/IIa on platelet adhesion in the presence of 2 mmol/L Mg2+ or Ca2+. Platelets were allowed to adhere (100x106/mL, 30 minutes, 22°C, stationary) on both PC (solid bars) (A) and NC (hatched bars) (B) in the presence or absence of 6F1 (10 µg/mL) or Gi9 (20 µg/mL). Data represent mean±SD; n=4 (Mg2+) and n=3 (Ca2+); *P<0.001; **P<0.0001.

SEM revealed interesting cation-dependent aspects of platelet morphology and confirmed that the selected assay conditions favored adhesion of single platelets (Figure 6Down). In the presence of Mg2+, PC- and NC-adherent platelets appeared similar, showing many pseudopods and full spreading of the cytoplasm (Figure 6Down, A and C). In the presence of Ca2+, only a few platelets adhered on PC, and they retained a round shape with a few projections (Figure 6BDown). In contrast, platelets on NC projected pseudopods and were still able to spread (Figure 6DDown). In addition, a notable proportion of "spongy"-appearing platelet ghosts was detected, which had swollen and no longer remained spread or had pseudopods.



View larger version (121K):
[in this window]
[in a new window]
  		Figure 6. SEMs illustrating the effects of the proteolysis of collagen substrate and divalent cations on the morphology of adherent GFPs (100x106/mL, 30 minutes, 22°C, stationary): adherent GFPs on PC in the presence of Mg2+ (A) and Ca2+ (B) and on NC in the presence of Mg2+ (C) and Ca2+ (D). Bar=1 µm. In the presence of Mg2+, platelets spread extensively independent of the substrate. In the presence of Ca2+, the few platelets on PC remained round with few pseudopods, whereas on NC, platelets spread and formed ruffled surfaces. On NC, there were also several sponge-like structures (arrow). This morphological feature was absent from platelets adherent on either NC or PC in the presence of Mg2+.

The adhesion results confirmed the blood flow studies in that GP Ia/IIa was essential for primary platelet adhesion to PC, whereas it was not mandatory for NC. Unlike PC, NC possessed the ability for adhesion and activation-related morphological changes in the presence of Ca2+.

Aggregation Studies
Because of the better aggregate-forming capacity of NC during blood flow, platelet aggregation was studied with PPACK-treated and citrated PRP and GFPs in the presence of divalent cations. Here, the platelet preference for NC over PC was also more obvious in citrated PRP, in which a 4-fold higher concentration of PC was needed to induce maximal aggregation of an extent similar to that with NC, but the lag time still remained twice as long (Table 1Down). In PPACK PRP, the 4-fold higher concentration of PC resulted in a compatible lag time, rate, and maximal aggregation. Although during the NC-induced aggregation the lag time did not vary with the anticoagulation, the rate was enhanced, benefiting from the presence of cations. The same cation-dependent enhancement was observed with PC. To exclude the role of fibril formation in the extended lag time, PC was preincubated at 35°C for 2 hours in a neutralizing buffer. Although this treatment improved the aggregating capacity of PC, the lag time, rate, and maximal aggregation all failed to match aggregation induced by the corresponding concentrations of NC (data not shown). 6F1 affected PC significantly more than NC by prolonging lag time and reducing the rate and maximal aggregation in citrated PRP, and these effects were even more pronounced in PPACK PRP (data not shown). The aggregation data of GFPs were complementary to those of PRP (Table 2Down). The aggregating capacity of PC was severely impaired when Mg2+ was omitted and Ca2+ was present. This also prolonged the lag time of NC-triggered aggregation, but once the aggregation started, its rate and maximum were the same irrespective of the ionic environment. In contrast to plasma milieu, fibril-forming treatment improved the PC-induced aggregation response of GFPs, especially in the presence of Mg2+ (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Comparison of NC- and PC-Induced Aggregation in Citrated and PPACK-Treated PRP


View this table:
[in this window]
[in a new window]
  		Table 2. Comparison of NC- and PC-Induced Aggregation of GFP in the Presence or Absence of Mg2+/Ca2+

Studies on Tyrosine Phosphorylation as a Marker of Activation
Time-dependent tyrosine phosphorylation was also assessed to capture the differential NC- and PC-induced activation of platelets. To discard the phosphorylation events dependent on GP IIb/IIIa activation, an inhibitory peptide (KQAGDV) was used. In the presence of Mg2+, both NC and prefibrillated PC induced similar patterns of tyrosine-phosphorylated proteins, but with NC, the kinetics of phosphorylation was slightly faster than with PC (Figure 7Down). The differences in phosphorylation patterns were accentuated in the presence of Ca2+, when several bands were not visible during PC induction or appeared only at the latest time points. The switch of cations also changed the NC-induced phosphorylation: in the presence of Ca2+, several bands appeared slowly or their intensity decreased.



View larger version (64K):
[in this window]
[in a new window]
  		Figure 7. Time-dependent (0, 15, 30, and 45 seconds and 1, 3, and 5 minutes) tyrosine phosphorylation patterns induced by stimulation of GFPs with 1.5 µg/mL of NC and of prefibrillated PC in the presence of 2 mmol/L Mg2+ (A) or Ca2+ (B) and KQAGDV. The control samples were stirred at 37°C for 1 minute. Example is representative of 3 separate donors.

Studies of Procoagulant Activity on Collagen-Adherent Platelets
Finally, the procoagulant activity of NC- and PC-adherent platelets was assessed by measurement of thrombin formation. Platelets adhering on NC had consistently increased procoagulant capacity compared with platelets adhering on PC, and platelets, which originally adhered to NC in the presence of Ca2+, generated the most thrombin (Figure 8Down). However, if the GFPs were first allowed to adhere in the presence of Mg2+ and the procoagulant activity was then developed by the addition of external Ca2+, significant thrombin formation was observed on PC, albeit still less than on NC when related to the number of bound platelets. Thrombin formation (µU · 106 GFPs-1 · min-1) was 11±4 (PC, Mg2+) <18±11 (NC, Mg2+) < 30±15 (NC, Ca2+), n=4.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 8. Procoagulant activity generated by platelets that had first adhered on NC (hatched bars) or PC (solid bars) in the presence of 2 mmol/L Mg2+ or Ca2+. Thrombin-forming capacity was assessed in the presence of diluted plasma and tissue thromboplastin. Samples of supernatants were reacted with the chromogenic thrombin substrate S-2238, and their absorbance was measured at 405 nm. A reagent control was subtracted from the values. Data represent mean±SD; n=4; *P<=0.01.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Structural differences resulting from the removal of collagen telopeptides and native quaternary structure (Figure 1Up) lead to reduced activation potential of the collagen, whereas the adhesive capacity remained, as illustrated by relatively similar surface coverages on both the collagen fibrils and monomers during perfusion (Figures 2Up and 3Up) (except at the high shear rate in citrated blood) and adhesion experiments with GFPs in the presence of Mg2+ cations (Figure 4Up). As previously reported, adhesion to monomeric collagen (1) was Mg2+-dependent and mediated by GP Ia/IIa,10 (2) was inhibited by Ca2+ cations,10 29 and (3) resulted in complete spreading of platelets41 (Figures 4Up, 5Up, and 6Up).

Our most interesting finding was the complete cation-dependence of the shear force–resistant thrombus formation on PC, in contrast to NC, which were capable of inducing thrombus formation cation-independently. Compatible with our other findings, this result manifests the importance of GP Ia/IIa as a mandatory primary receptor of platelet interaction with the collagen triple helix, as also reported by others.42 In support of our results, it was recently highlighted that GP Ia/IIa is irreplaceable in resisting shear forces.20 43 Being a poor adhesive substrate for GFPs in the presence of Ca2+ or in the absence of divalent cations in blood flowing at the high shear rate, monomers revealed the importance of the first collagen-adherent platelets in thrombus formation.37 Collagen monomers may offer a way to study the role and donor variability of GP Ia/IIa, which are likely to be associated with certain receptor polymorphisms and even thrombotic events.44 45

The differences between native-type collagen fibrils and monomers cannot be explained by differential vWF-binding capacity, because (1) vWF binding to collagen has been reported to be cation-independent,46 (2) the vWF-dependent increase in platelet deposition was observed on both substrates in PPACK-anticoagulated blood, (3) it was similarly inhibited by ATA,39 and (4) incubation of the monomer surface with vWF before perfusion (citrated blood, high shear rate) was unable to correct for the lacking platelet deposition, in contrast to restoration of the physiological Mg2+ concentration.

Adhesion to monomers correlated strongly with adhesion to fibrils in the presence of Mg2+, whereas Mg2+-dependent adhesion on monomers did not correlate with adhesion on fibrils in the presence of Ca2+, suggesting that at least 2 different receptors mediate platelet adhesion to collagen, depending on the experimental conditions and the substrate. Existence of additional collagen receptor(s) capable of replacing GP Ia/IIa was further supported by the ability of collagen fibrils to withhold thrombus formation under the high shear force (Figures 2Up and 3Up) and the unchanged adhesion under the conditions in which GP Ia/IIa–inhibiting antibodies and Ca2+ cations were present (Figures 4Up and 5Up). In the presence of GP Ia– and GP IV–blocking antibodies, {approx}50% of the platelet deposition remained in citrated blood at high shear rate, compatible with an unknown platelet adhesion receptor for native-type collagen fibrils.

In addition to containing the molecular determinants for GP Ia/IIa– and GP IV–independent adhesion, collagen fibrils induced platelet activation more efficiently than monomers, as also recently suggested by increased thromboxane A2 production triggered with acid-insoluble collagen.14 The better platelet-binding and aggregate-forming capacity and independence of divalent cations of fibrils observed during perfusions were supported by the aggregation studies, in which >=4 times fewer native-type fibrils than collagen monomers were needed, as reported previously.47 The morphology of adherent GFPs suggested that collagen fibrils activated platelets, in contrast to monomers (Figure 6Up). The receptor(s) using fibrils benefited from the presence of Ca2+ and the conditions that favored platelet-platelet interaction and activation (PPACK, high shear, 37°C). In the presence of Ca2+, augmented platelet activation by fibrils was also evidenced by the tyrosine-phosphorylation events (Figure 7Up) and enhanced procoagulant capacity (Figure 8Up). Conversely, the similarities in tyrosine phosphorylation induced by NC and PC in the presence of Mg2+ and the distinct NC-induced tyrosine phosphorylation patterns in different cationic milieus (Figure 7Up), as well as the equal procoagulant responses of NC and PC if the platelets were first allowed to bind in the presence of Mg2+ (Figure 8Up), all suggest that GP Ia/IIa also participates in platelet activation.48 49 Recently, the interplay in the GP Ia/IIa–GP IIb/IIIa axis was disturbed by mast cell–derived heparin proteoglycans, which strongly inhibited aggregate formation subsequent to adhesion on collagen.50

The identity of the hypothetical collagen receptor(s) acting on determinants on native-type fibrils was not elucidated by these experiments. The activating receptor is likely to be GP VI, which is reported to induce potent signal transduction by collagen51 52 or by agents acting solely through it.53 54 In this study, GP VI may participate, because NC induced faster tyrosine phosphorylation patterns, as well as a procoagulant response,55 which could accord with the morphological changes of platelet deposition on NC in the presence of Ca2+ (Figure 6Up). However, it has been reported that under blood flow, GP VI alone is incapable of retaining adhesion, so it is unlikely that it acts as a primary adhesive receptor.3 20 56 We found that in citrated blood, an mAb against GP IV (131.7) was able to inhibit platelet deposition on NC in the early adhesive phase by 49%, as reported previously.36 Thus, it is possible that when GP Ia/IIa and GP IV are eliminated in our perfusion experiments and when vWF is unable to firmly stop platelets, a third, as yet unidentified receptor plays a necessary part in the early platelet interaction with native collagen fibrils.

In the light of the present understanding of platelet-collagen interaction,3 5 57 our results suggest that (1) GP Ia/IIa is an absolute requirement for the collagen triple helix to support shear-resistant thrombus growth, and thus monomers provide a tool to study GP Ia/IIa activity in individual donors; (2) the native fibril structure of the collagen molecule is involved in platelet-activating events such as aggregate formation, tyrosine phosphorylation signaling, and development of procoagulant activity; (3) an unknown receptor acting via quaternary structure/telopeptides is capable of replacing GP IV–, GP Ia/IIa–, and cation-dependent functions in primary adhesion; and (4) when functional, GP Ia/IIa also participates in the events leading to platelet activation.


*    Acknowledgments
 
The authors acknowledge the excellent technical assistance of Marja Lemponen and Tuula Järvenpää. We thank Dr Matti Vauhkonen for assistance in the collagen preparations and Dr Michael Hess (Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki) for the TEM micrographs. While this work was in press, related findings were published: Savage B, Glusberg MH, Ruggeri ZM. Influence of fibrillar collagen structure on the mechanisms of platelet thrombus formation under flow. Blood. 1999;94:2704–2715.

Received February 3, 1999; accepted March 19, 1999.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Baumgartner HR. Platelet interaction with collagen fibrils in flowing blood: reaction of human platelets with {alpha}-chymotrypsin-digested subendothelium. Thromb Haemost. 1977;37:1–16.[Medline] [Order article via Infotrieve]
  2. Kehler B. Platelet receptors for collagen. Platelets. 1995;6:11–16.
  3. Sixma JJ, van Zanten H, Huizinga EG, van der Plas RM, Verkleij M, Wu Y-P, Gros P, de Groot PG. Platelet adhesion to collagen: an update. Thromb Haemost. 1997;78:434–438.[Medline] [Order article via Infotrieve]
  4. Heemskerk JWM, Vuist WMJ, Feijge MAH, Reutelingsperger CPM, Lindhout T. Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses. Blood. 1997;90:2615–2625.[Abstract/Free Full Text]
  5. Moroi M, Jung SM. Platelet receptors for collagen. Thromb Haemost. 1997;78:439–444.[Medline] [Order article via Infotrieve]
  6. Siegel BM, Friedman IA, Schwartz SO. Hemorrhagic disease in osteogenesis imperfecta: study on platelet functional defect. Am J Med. 1957;22:315–322.[Medline] [Order article via Infotrieve]
  7. Johnson GJ, Holloway DE, Hutton SW, Duane WC. Platelet function in scurvy and experimental human vitamin C deficiency. Thromb Res. 1981;24:85–96.[Medline] [Order article via Infotrieve]
  8. Hindriks GA, Sixma JJ, de Groot PG. Ascorbic acid increases thrombogenicity of cellular matrices. Thromb Haemost. 1991;66:505–509.[Medline] [Order article via Infotrieve]
  9. Nieuwenhuis HK, Akkerman JWN, Houdijk WPM, Sixma JJ. Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia. Nature. 1985;318:470–472.[Medline] [Order article via Infotrieve]
  10. Santoro SA. Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen. Cell. 1986;46:913–920.[Medline] [Order article via Infotrieve]
  11. Kunicki TJ, Nugent DJ, Staats SJ, Orchekowski RP, Wayner EA, Carter WG. The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex. J Biol Chem. 1988;263:4516–4519.[Abstract/Free Full Text]
  12. Saelman EUM, Kehler 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.[Abstract/Free Full Text]
  13. Diaz-Ricart M, Tandon NN, Gomez-Ortiz G, Carretero M, Escolar G, Ordinas A, Jamieson GA. Antibodies to CD36 (GPIV) inhibit platelet adhesion to subendothelial surfaces under flow conditions. Arterioscler Thromb Vasc Biol. 1996;16:883–888.[Abstract/Free Full Text]
  14. Nakamura T, Jamieson GA, Okuma M, Kambayashi J, Tandon NN. Platelet adhesion to native type I collagen fibrils: role of GPVI in divalent cation-dependent and -independent adhesion and thromboxane A2 generation. J Biol Chem. 1998;273:4338–4344.[Abstract/Free Full Text]
  15. Kotite NJ, Cunningham LW. Specific adsorption of a platelet membrane glycoprotein by human insoluble collagen. J Biol Chem. 1986;261:8342–8347.[Abstract/Free Full Text]
  16. Deckmyn E, van Houtte E, Vermylen J. Disturbed platelet aggregation to collagen associated with an antibody against an 85/90 kD platelet glycoprotein in a patient with prolonged bleeding time. Blood. 1992;79:1466–1471.[Abstract/Free Full Text]
  17. Chiang TM, Rinaldy A, Kang AH. Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen. J Clin Invest. 1997;100:514–521.[Medline] [Order article via Infotrieve]
  18. Morton LF, Hargreaves PG, Farndale RW, Young RD, Barnes MJ. Integrin {alpha}2ß1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for {alpha}2ß1-independent platelet reactivity. Biochem J. 1995;306:337–344.
  19. Barnes MJ, Knight CG, Farndale RW. Collagens and atherosclerosis: cell-collagen interaction. In: Jacotot B, Mathé D, Fruchart J-C, eds. Atherosclerosis XI. Singapore: Elsevier Science; 1998:299–306.
  20. Verkleij MW, Morton LF, Knight CG, de Groot PG, Barnes MJ, Sixma JJ. Simple collagen-like peptides support platelet adhesion under static but not under flow conditions: interaction via {alpha}2ß1 and von Willebrand factor with specific sequences in native collagen is a requirement to resist shear forces. Blood. 1998;91:3808–3816.[Abstract/Free Full Text]
  21. Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochem J. 1996;316:1–11.
  22. Leibovich SJ, Weiss JB. Electron microscope studies of the effects of endo- and exopeptidase digestion on tropocollagen: a novel concept of the role of terminal regions in fibrillogenesis. Biochim Biophys Acta. 1970;214:445–454.[Medline] [Order article via Infotrieve]
  23. Saelman UM, Morton LF, Barnes MJ, Gralnick HR, Hese KM, Nieuwenhuis HK, de Groot PG, Sixma JJ. Platelet adhesion to cyanogen-bromide fragments of collagen {alpha}1(I) under flow conditions. Blood. 1993;82:3029–3033.[Abstract/Free Full Text]
  24. Morton LF, Peachey AR, Barnes MJ. Platelet-reactive sites in collagens type I and III: evidence for separate adhesion and aggregatory sites. Biochem J. 1989;258:157–163.[Medline] [Order article via Infotrieve]
  25. Santoro SA, Walsh JJ, Staatz WD, Baranski KJ. Distinct determinants on collagen support {alpha}2ß1 integrin-mediated platelet adhesion and platelet activation. Cell Regul. 1991;2:905–913.[Medline] [Order article via Infotrieve]
  26. Miller EJ, Rhodes RK. Preparation and characterization of the different types of collagen. Methods Enzymol. 1982;82:36–64.
  27. Kivirikko KI, Laitinen O, Prockop DJ. Modifications of a specific assay for hydroxyproline in urine. Anal Biochem. 1967;19:249–255.[Medline] [Order article via Infotrieve]
  28. Sakariassen KS, Muggli R, Baumgartner HR. Measurement of platelet interaction with components of the vessel wall in flowing blood. Methods Enzymol. 1989;169:37–70.[Medline] [Order article via Infotrieve]
  29. Grzesiak JJ, Davis GE, Kirchhofer D, Pierschbacher MD. Regulation of {alpha}2ß1-mediated fibroblast migration on type I collagen by shifts in the concentrations of extracellular Mg2+ and Ca2+. J Cell Biol. 1992;117:1109–1117.[Abstract/Free Full Text]
  30. Timmons S, Hawiger J. The isolation of platelets by albumin gradient and gel filtration. Methods Enzymol. 1989;169:11–22.[Medline] [Order article via Infotrieve]
  31. Mustonen P, Lassila R. Epinephrine augments platelet recruitment to immobilized collagen in flowing blood: evidence for a von Willebrand factor-mediated mechanism. Thromb Haemost. 1996;75:175–181.[Medline] [Order article via Infotrieve]
  32. Holmes DF, Capaldi MJ, Chapman JA. Reconstitution of collagen fibrils in vitro: the assembly process depends on the initiating procedure. Int J Biol Macromol. 1986;8:161–166.
  33. Badimon L, Badimon JJ, Galvez A, Fuster V. Influence of arterial damage and wall shear rate on platelet deposition. Arteriosclerosis. 1986;6:312–320.[Abstract/Free Full Text]
  34. Lassila R, Badimon JJ, Vallabhajosula S, Badimon L. Dynamic monitoring of platelet deposition on severely damaged vessel wall in flowing blood. Arteriosclerosis. 1990;16:306–315.
  35. 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/IIIa mediated by adhesive proteins. Blood. 1989;74:182–192.[Abstract/Free Full Text]
  36. Matsuno K, Diaz-Ricart M, Montgomery RR, Ashter RH, Jamieson GA, Tandon NN. Inhibition of platelet adhesion to collagen by monoclonal anti-CD36 antibodies. Br J Haematol. 1996;92:960–967.[Medline] [Order article via Infotrieve]
  37. Siljander P, Carpén O, Lassila R. Platelet-derived microparticles associate with fibrin during thrombosis. Blood. 1996;87:4651–4663.[Abstract/Free Full Text]
  38. Kunicki TJ, Orchekowski R, Annis D, Honda Y. Variability of integrin {alpha}2ß1 activity on human platelets. Blood. 1993;82:2693–2703.[Abstract/Free Full Text]
  39. Phillips MD, Moake JL, Nolasco L, Turner N. Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets. Blood. 1988;72:1898–1903.[Abstract/Free Full Text]
  40. Staatz WD, Peters KJ, Santoro SA. Divalent cation-dependent structure in the platelet membrane glycoprotein Ia-IIa (VLA-2) complex. Biochem Biophys Res Commun. 1990;168:107–113.[Medline] [Order article via Infotrieve]
  41. Nieuwenhuis HK, Sakariassen KS, Houdijk WPM, Nievelstein PFEM, Sixma JJ. Deficiency of platelet membrane glycoprotein Ia associated with a decreased platelet adhesion to subendothelium: a defect in platelet spreading. Blood. 1986;68:692–695.[Abstract/Free Full Text]
  42. Saelman EUM, Nieuwenhuis HK, Hese KM, de Groot PG, Heijnen HFG, Sage EH, Williams S, McKeown L, Gralnick HR, Sixma JJ. Platelet adhesion to collagen types I through VIII under conditions of stasis and flow is mediated by GPIa/IIa ({alpha}2ß1-integrin). Blood. 1994;83:1244–1250.[Abstract/Free Full Text]
  43. Tanaka M, Handa M, Kawakami K, Kokubum T, Oda A, Ohta Y, Kudoh S, Ikeda Y. {alpha}2ß1 integrin and GPIb/IX complex engagements in platelet adhesion to type I collagen under flow conditions. Blood. 1997;90:27a. Abstract.
  44. Kritzik M, Savage B, Nugent DJ, Santoso S, Ruggeri ZM, Kunicki TJ. Nucleotide polymorphism in the alpha 2 gene define multiple alleles that are associated with differences in platelet alpha 2 beta 1 density. Blood. 1998;92:382–388.
  45. Moshfegh K, Wuillemin WA, Redondo M, Lämmle B, Beer JH, Liechti-Gallati S, Meyer BJ. Association of two silent polymorphisms of platelet glycoprotein Ia/IIa receptor with risk of myocardial infarction: a case-control study. Lancet. 1999;353:351–354.[Medline] [Order article via Infotrieve]
  46. Usui T, Fujisawa T, Takagi J, Saito Y. Propolypeptide and mature portions of von Willebrand factor of bovine origin recognize different sites on type-I collagen obtained from bovine tendon. Eur J Biochem. 1992;205:363–367.[Medline] [Order article via Infotrieve]
  47. Csako G, Suba EA, Herp A. Monomeric and polymeric collagen-induced platelet aggregation in citrated and heparinized platelet-rich plasma. Connect Tissue Res. 1985;13:299–311.[Medline] [Order article via Infotrieve]
  48. Keely PJ, Parise LV. The {alpha}2ß1 integrin is a necessary co-receptor for collagen-induced activation of Syk and the subsequent phosphorylation of phospholipase C{gamma}2 in platelets. J Biol Chem. 1996;271:26668–26676.[Abstract/Free Full Text]
  49. Kamiguti AS, Markland FS, Zhou Q, Laing GD, Theakston RGD, Zuzel M. Proteolytic cleavage of the ß1 subunit of platelet {alpha}2ß1 integrin by the metalloproteinase jararhagin compromises collagen-stimulated phosphorylation of pp72syk. J Biol Chem. 1997;272:32599–32605.[Abstract/Free Full Text]
  50. Lassila R, Lindstedt K, Kovanen PT. Native macromolecular heparin proteoglycans exocytosed from stimulated rat serosal mast cells strongly inhibit platelet-collagen interactions. Arterioscler Thromb Vasc Biol. 1997;17:3578–3587.[Abstract/Free Full Text]
  51. Ichinohe T, Takayama H, Ezumi Y, Arai M, Yamamoto N, Takahashi H, Okuma M. Collagen-stimulated activation of Syk but not c-Src is severely compromised in human platelets lacking membrane glycoprotein VI. J Biol Chem. 1997;272:63–68.[Abstract/Free Full Text]
  52. Kehrel B, Wierwille S, Clemetson KJ, Anders O, Steiner M, Knight CG, Farndale RW, Okuma M, Barnes MJ. Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not. Blood. 1998;91:491–499.[Abstract/Free Full Text]
  53. Achison M, Joel C, Hargreaves PG, Sage SO, Barnes MJ, Farndale RW. Signal elicited from human platelets by synthetic, triple helical, collagen-like peptides. Blood Coagul Fibrinolysis. 1996;7:149–152.[Medline] [Order article via Infotrieve]
  54. Polgar J, Clemetson JM, Kehrel BE, Wiedemann M, Magnenat EM, Wells TNC, Clemetson KJ. Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. J Biol Chem. 1997;272:13576–13583.[Abstract/Free Full Text]
  55. Heemskerk JWM, Siljander P, Vuist WMJ, Breikers G, Reutelingsperger CPM, Barnes MJ, Knight CG, Lassila R, Farndale RW. Function of glycoprotein VI and integrin {alpha}2ß1 in the procoagulant response of single, collagen-adherent platelets. Thromb Haemost.. 1999;81:782–792.[Medline] [Order article via Infotrieve]
  56. Moroi M, Jung SM, Shinmyozu K, Tomiyama Y, Ordinas A, Diaz-Ricart M. Analysis of platelet adhesion to a collagen-coated surface under flow conditions: the involvement of glycoprotein VI in the platelet adhesion. Blood. 1996;88:2081–2092.[Abstract/Free Full Text]
  57. Barnes MJ, Knight CG, Farndale RW. The collagen-platelet interaction. Curr Opin Hematol. 1998;5:314–320.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 BloodHome page
S. Yoshida, T. Sudo, M. Niimi, L. Tao, B. Sun, J. Kambayashi, H. Watanabe, E. Luo, and H. Matsuoka
Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito
Blood, February 15, 2008; 111(4): 2007 - 2014.
[Abstract] [Full Text] [PDF]