In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking a2b1 mAb, abro-gated platelet adhesion to fibrillar and soluble collagen.. Additionally, sara-tin
Trang 1integrin a2b1 and von Willebrand factor binding to collagen Tara C White1, Michelle A Berny1, David K Robinson1, Hang Yin2, William F DeGrado2,3,
Stephen R Hanson1and Owen J T McCarty1,4
1 Department of Biomedical Engineering, Oregon Health & Science University, Portland, OR, USA
2 Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
3 Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA
4 Cell and Developmental Biology, Oregon Health & Science University, Portland, OR, USA
Collagen plays a critical role in mediating the platelet
response to vessel injury in the dynamic environment
of the vasculature Exposed collagen at sites of
vascu-lar injury initiates two platelet functions fundamental
to the process of primary hemostasis: initial
recruit-ment of circulating platelets, and triggering of the
platelet activation cascade required to stimulate
throm-bus growth [1,2] The first step in platelet recruitment
to collagen occurs indirectly, via binding of platelet glycoprotein (GP)Ib to collagen-bound von Willebrand factor (VWF) [3] VWF plays a critical role in the teth-ering of platelets at high shear levels, due to the rapid on-rate of binding between GPIb and VWF The rapid off-rate of GPIb–VWF interactions results in platelet
Keywords
a2b1; collagen; platelet; saratin;
von Willebrand factor
Correspondence
O J T McCarty, Department of Biomedical
Engineering, Oregon Health & Science
University, 13B-CHH, 3303 SW Bond Ave,
Portland, OR 97239, USA
Fax: +1 503 418 9311
Tel: +1 503 418 9307
E-mail: mccartyo@ohsu.edu
(Received 22 October 2006, revised 20
December 2006, accepted 11 January 2007)
doi:10.1111/j.1742-4658.2007.05689.x
Subendothelial collagen plays an important role, via both direct and indir-ect mechanisms, in the initiation of thrombus formation at sites of vascular injury Collagen binds plasma von Willebrand factor, which mediates plate-let recruitment to collagen under high shear Subsequently, the direct bind-ing of the platelet receptors glycoprotein VI and a2b1to collagen is critical for platelet activation and stable adhesion Leeches, have evolved a number
of inhibitors directed towards platelet–collagen interactions so as to prevent hemostasis in the host during hematophagy In this article, we describe the molecular mechanisms underlying the ability of the leech product saratin
to inhibit platelet binding to collagen In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking a2b1 mAb, abro-gated platelet adhesion to fibrillar and soluble collagen Additionally, sara-tin eliminated a2b1-dependent platelet adhesion to soluble collagen in the presence of an Src kinase inhibitor Moreover, saratin prevented platelet-rich plasma adhesion to fibrillar collagen, a process dependent upon both
a2b1 and von Willebrand factor binding to collagen Furthermore, saratin specifically inhibited the binding of the a2integrin subunit I domain to col-lagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of a combination of mAbs to glycopro-tein Ib and a2b1 These results demonstrate that saratin interferes with inte-grin a2b1 binding to collagen in addition to inhibiting von Willebrand factor–collagen binding, presumably by binding to an overlapping epitope
on collagen This has significant implications for the use of saratin as a tool to inhibit platelet–collagen interactions
Abbreviations
a 2 I-bio, biotinylated a 2 integrin subunit I domain; DIC, differential interference contrast; FITC, fluorescein isothiocyanate; GP, glycoprotein; PRP, platelet-rich plasma; TxA2, thromboxane A2; VWF, von Willebrand factor.
Trang 2translocation at the site of injury, allowing adhesive
interactions with slower binding kinetics (such as the
platelet collagen receptors GPVI and a2b1 and aIIbb3
integrins) to mediate platelet adhesion and activation
[4] Two routes have been proposed for this second
step of platelet adhesion, namely GPVI-mediated
platelet activation either preceding or following a2b1
integrin-mediated platelet adhesion [5,6] It is
notewor-thy that under static or low-shear conditions, the roles
of VWF and GPIb are dispensable, as the collagen
receptors GPVI and a2b1can mediate platelet adhesion
independently of VWF
The evolution of a panoply of molecules to
inter-fere with the process of hemostasis has allowed the
leech to continue its alimentary habit of
hemato-phagy The presence of anticoagulants in the salivary
glands of the leech, Hirudo medicinalis, was originally
discovered by Haycraft in 1884 and led to the
isola-tion of hirudin, a potent antithrombin anticoagulant
[7] In addition to molecules that target the
coagula-tion cascade, a number of leech-derived substances
have been discovered that inhibit platelet adhesion
and activation Three such molecules, LAPP (an
approximately 13 kDa leech antiplatelet protein
isola-ted from Haementeria officinalis) and calin and
sara-tin (approximately 65 kDa and 12 kDa proteins,
respectively, both isolated from H medicinalis), have
been shown to specifically block platelet–collagen
interactions by inhibiting VWF binding to collagen
[8–11] Depraetere et al [12] then went on to
demon-strate that both LAPP and calin block the binding
site on collagen for the platelet integrin a2b1 The
saratin-binding site on collagen responsible for the
inhibition of VWF binding is presently unknown
Saratin, which consists of 103 amino acids and
con-tains three disulfide bridges [13], has been cloned and
produced in recombinant form in Hansenula
polymor-pha Barnes et al were the first to demonstrate that
saratin specifically blocks purified VWF binding to
col-lagen, as well as potently inhibiting platelet aggregate
formation on immobilized collagen under shear flow
[8], therefore leading to the extensive use in the
litera-ture of saratin as a VWF–collagen inhibitor [14–18]
Furthermore, saratin has been shown to inhibit lumen
stenosis in carotid endarterectomized rats [19] and to
reduce platelet adhesion and intimal hyperplasia in
both a nondiseased environment [20] and in the state
of hyperhomocystinemia [21] Moreover, Vilahur et al
demonstrated that local administration of saratin
inhibited atherosclerotic plaque thrombogenicity under
shear conditions [22]
The main collagen-binding site on VWF resides
within the A3 domain (residues 923–1109) of VWF
[23–25] Structural studies on the VWF A3 domain showed that it assumes the same fold as the binding site for collagen on the a2b1 integrin, namely the homologous integrin a2 I domain [26] The present study demonstrates that saratin interferes with integrin
a2b1 binding to collagen, in addition to inhibiting VWF–collagen binding, presumably by binding to an overlapping epitope on collagen This has significant implications for the use of saratin as a tool to inhibit platelet–collagen interactions, and may provide the basis for the therapeutic use of saratin as a potent antithrombotic agent
Results
Delayed collagen-induced aggregation
of platelets in the presence of saratin
We initially investigated the effects of the leech prod-uct saratin on the ability of platelets to aggregate in response to fibrillar collagen Consistent with previous findings [8], dose–response and maximal aggregation
of platelets did not differ in the presence of saratin (data not shown) However, onset of aggregation was significantly delayed in the presence of saratin (Fig 1A), and this lag time was particularly evident
at low fibrillar collagen concentrations (Fig 1B) Moreover, a similar delay in collagen-induced aggre-gation was observed in the presence of the a2b1 -blocking antibody 6F1 (data not shown), consistent with previous reports demonstrating an a2b1 -depend-ent lag phase for collagen-induced aggregation [29] Together, these findings led us to question whether saratin blocks platelet a2b1 binding to collagen in addition to functioning as an inhibitor of VWF– collagen binding, as had been previously described by Barnes et al [8]
Dissection of the molecular actions of saratin
on fibrillar collagen Experiments were designed to evaluate the ability of saratin to inhibit platelet adhesion to collagen We gently pipetted purified human platelets onto surface-immobilized fibrillar collagen, and recorded the degree
of adhesion and spreading using Normarski differential interference contrast (DIC) microscopy In agreement with previous reports, human platelets undergo com-plete spreading on fibrillar collagen in the absence of external stimulation (Fig 2A) The degree of platelet adhesion to fibrillar collagen was only slightly reduced
by the presence of either an a2b1-blocking antibody or saratin; however, these effects were statistically
Trang 3insigni-ficant (Fig 2B) In comparison, a 40% reduction in
the degree of platelet adhesion to fibrillar collagen was
observed in the presence of apyrase and indomethacin,
inhibitors of the secondary mediators ADP and
throm-boxane A2(TxA2), respectively (Fig 2B) Furthermore,
the integrin a2b1mediates this ADP⁄ TxA2-independent
platelet adhesion to fibrillar collagen, as evidenced by
the abrogation of platelet binding in the presence of the
a2b1mAb 6F1 (Fig 2A) Along these lines, ADP⁄ TxA2
-independent platelet adhesion to fibrillar collagen was
eliminated in the presence of saratin (Fig 2A)
Import-antly, saratin was not required to be present in
suspen-sion to have an inhibitory effect, as pretreatment of the collagen surface with saratin was sufficient to achieve blockade of platelet adhesion in the presence
of apyrase⁄ indomethacin (Table 1) In contrast, 6F1 needed to be present in the suspension to achieve blockade (Table 1) However, it is noteworthy that the inhibition of platelet adhesion observed in the presence
of 6F1 or saratin in the absence of secondary media-tors could be overcome by treatment of platelet sus-pensions with the G protein-coupled receptor agonist thrombin (Fig 2C,D)
Dissection of the molecular actions of saratin
on soluble collagen
We next aimed to examine platelet attachment to sol-uble collagen, a process that has been reported to be predominately mediated via a2b1 integrins [31,32] Indeed, the presence of the a2b1 mAb 6F1 reduced platelet adhesion on soluble collagen by over 60% (Fig 3A,B) Along these lines, a similar degree of inhi-bition was observed in the presence of saratin (Fig 3B)
We extended our studies to examine the effects of secondary mediators on platelet adhesion to soluble collagen In parallel with our observations on fibrillar collagen, a 50% reduction in platelet adhesion on soluble collagen was observed in the presence of the ADP scavenger apyrase and the cyclooxygenase inhib-itor indomethacin (Fig 3B) Moreover, ADP⁄ TxA2 -independent platelet adhesion to soluble collagen was eliminated through the blockade of a2b1 with 6F1 or treatment of collagen with saratin (Fig 3A) As was observed with fibrillar collagen, saratin did not need to
be present in suspension to have an inhibitory effect
on platelet adhesion (Table 1) However, in distinct contrast to what was observed with fibrillar collagen, both the a2b1 mAb 6F1 and saratin blocked thrombin-stimulated platelet adhesion to soluble collagen in the presence, but not the absence, of inhibitors of secon-dary mediators (Fig 3C,D)
It is noteworthy that the presence of saratin did not have any effect on platelet adhesion to immobilized fibrinogen (Table 1), indicating that saratin does not inhibit platelet integrin aIIbb3 binding to fibrinogen
Saratin blocks Src kinase-independent platelet adhesion to soluble collagen
A set of experiments was designed to investigate the role of Src family kinases in supporting platelet adhe-sion and spreading on soluble collagen As shown in Fig 4B, a 40% reduction in the degree of adhesion was observed in the presence of the Src kinase
inhib-Fig 1 The effect of saratin on the time of onset (lag phase) of
shape change in response to fibrillar collagen (A) Heparinized
human PRP was stimulated with different concentrations of fibrillar
collagen in the absence or presence of saratin (10 lgÆmL)1)
Chan-ges in attenuance indicative of shape change and aggregation were
recorded (B) The delay in onset of platelet shape change (lag time)
in the absence (black bars) and presence (white bars) of saratin is
expressed as time (seconds) between addition of collagen and
ini-tial increase in the attenuance of the platelet suspension Values
are reported as follows: mean ± SEM from at least three
experi-ments *P < 0.05, d P < 0.01, with respect to vehicle-treated
sample.
Trang 4itor PP2, whereas platelets that bound to soluble
colla-gen in an Src kinase-independent manner were unable
to form lamellipodia Furthermore, the presence of the
a2b1 mAb 6F1 in combination with the Src kinase
inhibitor PP2 eliminated platelet adhesion to soluble
collagen altogether (Fig 4A) Importantly, saratin was
capable of blocking this Src kinase-independent
adhe-sion to soluble collagen (Fig 4A), consistent with the
ability of saratin to block a2b1-mediated platelet
bind-ing It is noteworthy that this series of experiments
was performed in the absence of inhibitors of ADP
and TxA2
Saratin blocks platelet-rich plasma adhesion
to collagen
Thus far, this study has utilized washed platelets in
order to examine the molecular mechanisms of saratin
Physiologically, however, platelets are exposed to
col-lagen in the presence of plasma proteins In order to
investigate the ability of saratin to inhibit
receptor-mediated interactions under physiologic conditions, we
layered platelet-rich plasma (PRP) over immobilized
collagen Our studies demonstrated that individual
platelets in citrated PRP bound to immobilized soluble collagen; however, interestingly, these platelets were unable to form lamellipodia (Fig 5A) Moreover, the presence of either the a2b1 mAb 6F1 or saratin abro-gated this adhesion (Fig 5A), further demonstrating the ability of saratin to block a2b1-mediated platelet binding Equivalent results were observed in PPACK⁄ heparin-anticoagulated PRP, which preserves the physiologic levels of divalent cations (36.2 ± 3.2 versus 0.74 ± 0.25· 10)2platelets⁄ mm2 on soluble collagen in the presence or absence of 10 lgÆmL)1 sar-atin, respectively; mean ± SEM; n¼ 3)
In contrast to studies using washed platelets, where
we found individual platelets to be adherent to fibrillar collagen, platelets in citrated PRP were incorporated into a fibrous mesh along the collagen fibres (Fig 5B) The degree of platelet⁄ fibrin deposition onto collagen fibres was unaffected by the presence of 6F1 (Fig 5B) However, the presence of saratin eliminated the ability
of PRP to form a fibrous mesh, and significantly reduced the degree of platelet adhesion to fibrillar colla-gen Importantly, we observed a similar level of reduc-tion in platelet⁄ fibrin deposition and platelet adhesion when the a2b1mAb 6F1 was used in combination with
Fig 2 The effect of saratin on platelet adhesion on immobilized fibrillar collagen Human washed platelets (2 · 10 7
mL)1) were placed on fibrillar collagen-coated cov-erslips for 45 min at 37 C, and imaged using DIC microscopy In selected experi-ments, the function-blocking a2b1mAb 6F1 (10 lgÆmL)1) or saratin (10 lgÆmL)1) was added to the platelet suspension, either in the absence (A, B) or the presence (C, D) of thrombin (1 UÆmL)1) Experiments were per-formed in the absence (black bars) or pres-ence (white bars) of the ADP-removing enzyme apyrase (apy) and the cyclooxyge-nase inhibitor indomethacin (indo) as indica-ted The numbers of adherent platelets were recorded for five fields of view (0.013 mm 2 ) and expressed as mean ± SEM from at least three experiments.
*P < 0.01 with respect to platelet adhesion
in the absence of apy ⁄ indo for each respec-tive treatment;dP < 0.01 with respect to platelet adhesion in the presence of apy ⁄ indo and absence of 6D1 or saratin.
Trang 5antagonists to VWF receptors on platelets, namely the
mAb to GPIb, 6D1, and the mAb to aIIbb3, LJ-CP8
Taken together, these data are reflective of the ability of
saratin to both block VWF–collagen binding and to
inhibit a2b1–collagen interactions
Inhibition of a2integrin subunit I domain binding
to collagen by saratin
In an attempt to determine whether the binding site on
collagen for the platelet receptor a2b1 is blocked by
the leech product saratin, we utilized a biotinylated
recombinant a2 integrin subunit I domain construct
Previous studies have shown that the a2 integrin
sub-unit I domain binds to collagen type I in a
dose-dependent and saturable manner [12] To investigate
the ability of saratin to inhibit a2b1 binding, coverslips
were coated with fibrillar collagen type I and
preincu-bated with or without saratin Subsequently, a
con-stant amount of biotinylated a2 integrin subunit I
domain (a2I-bio) was added, and the amount of a2
I-bio was detected by adding streptavidin–fluorescein
isothiocyanate (FITC) and visualized using fluores-cence microscopy Our results demonstrated that sara-tin was able to abrogate a2I-bio binding to collagen (Fig 6) In addition, saratin was able to completely block VWF binding to immobilized collagen (data not shown), consistent with previous reports [8,14] Taken together, our results definitively demonstrate that sara-tin potently inhibits a2b1 binding to collagen in addi-tion to blocking VWF–collagen interacaddi-tions
Saratin reduces platelet adhesion to collagen under flow conditions
We next aimed to examine the effects of saratin on platelet adhesion in a more physiologically relevant setting We therefore investigated platelet recruitment and aggregation as a result of the perfusion of whole blood at 1000 s)1 over immobilized fibrillar collagen
As shown in Fig 7, substantial platelet aggregates form on collagen under flow, producing 39.6 ± 1.9 thrombi per field of view, resulting in 34.7 ± 6.2% surface coverage (Table 2) Platelet adhesion was seve-rely reduced in the presence of the GPIb mAb 6D1, as evidenced by a dramatic reduction in surface coverage (Fig 7, Table 2) It is noteworthy that a number of thrombi consisting of one to three platelets were observed in the presence of 6D1 (Fig 7), whereas the number of these small thrombi was significantly reduced in the presence of the a2b1 mAb 6F1 in com-bination with the GPIb mAb 6D1 (Table 2) Import-antly, the presence of saratin reduced both the percentage of surface coverage and the amount of thrombi formed to a similar level as observed in the presence of the GPIb and a2b1 antagonists Similar results were observed in reconstituted blood (data not shown) Altogether, our results demonstrate that sara-tin, through blockade of both VWF and a2b1 binding
to collagen, acts as a potent inhibitor of platelet aggre-gation on collagen under shear flow conditions
Discussion
Previous studies have demonstrated that the leech product saratin functions as a potent inhibitor of VWF binding to collagen [8,14] In this study, we extend these findings to demonstrate that saratin addi-tionally functions as an inhibitor of platelet integrin
a2b1 binding to collagen This has important implica-tions for the interpretation of results obtained when saratin is used as an inhibitor of platelet–collagen interactions, both in vitro [14–18] and in vivo [19–22] The current study, in accordance with others [5,31,32], demonstrates that a2b1 integrins are not
Table 1 Effects of an a2b 1 blocker and saratin on platelet adhesion
on collagen Purified human platelets (2 · 10 7 mL)1), in the
pres-ence of apyrase (2 U mL)1) and indomethacin (10 l M ), were placed
on BSA, fibrillar or soluble collagen or fibrinogen-coated coverslips
for 45 min at 37 C In designated experiments, immobilized
colla-gen or fibrinocolla-gen was treated with the a2b1-blocking mAb 6F1
(10 lgÆmL)1) or saratin (10 lgÆmL)1) for 10 min, followed by
wash-ing with NaCl ⁄ P i , prior to exposure to platelets (surface treatment).
In selected experiments, 6F1 (10 lgÆmL)1) or saratin (10 lgÆmL)1)
was added to and maintained in the suspension with the platelets
throughout the adhesion assay (suspension treatment) Values are
reported as follows: adherent platelets, mean ± SEM of three to
six experiments; platelet surface area, mean ± SEM of 50–300
cells.
Surface
Surface treatment
Suspension treatment
Platelet adhesion (cells ⁄ mm 2 · 10)2)
*,**P < 0.01 with respect to platelet adhesion ⁄ surface area on
untreated fibrillar or soluble collagen, respectively.
Trang 6essential for platelet, whether purified or in plasma,
adhesion to fibrillar collagen, as GPVI is capable of
triggering platelet activation and release of secondary
mediators (ADP and TxA2), which leads to platelet
adhesion independently of a2b1 However, in the
absence of the actions of secondary mediators,
GPVI-mediated activation alone is insufficient to
induce platelet adhesion to fibrillar collagen in the
absence of a2b1, consistent with the current paradigm
[5,33,34]
A different picture emerges for platelet adhesion to
soluble collagen This form of collagen results from
the cleavage of collagen in the nontriple helical region,
where covalent cross-links are found that are required
for the assembly of collagen molecules into the typical
banded structure found in fibrillar collagen Soluble
collagen therefore lacks the highly repetitive GPVI
recognition sites characteristic of fibrillar collagen,
therefore providing a means of reducing but not
ablat-ing GPVI signalablat-ing [1,35] Consistent with previous
reports [32–34,36,37], our data demonstrate that a2b1
integrins play an important role in mediating platelet
adhesion to soluble collagen in the absence of
inhibi-tors of secondary mediainhibi-tors, whereas a2b1 is essential
for ADP⁄ TxA2-independent platelet adhesion Addi-tionally, we demonstrate that PRP binding to immobi-lized soluble collagen is a2b1-dependent Moreover, the
a2b1 dependency of Src kinase-independent adhesion
on soluble collagen further indicates the essential role
of a2b1 in the absence of platelet activation Interest-ingly, we found that thrombin stimulation, which pre-dominantly acts via the Gq family of proteins [38], potentiated a2b1-independent platelet adhesion to sol-uble collagen only in the presence of the actions of the
Gi protein-coupled agonist ADP This supports the notion that GPVI-mediated platelet activation and adhesion on the low-GPVI-affinity soluble collagen is dependent upon a cosignal from Gi-coupled receptors
in the absence of a2b1[37,39,40]
The ability of saratin to precisely mirror the effects of 6F1 in the aforementioned experiments, in combination with the fact that saratin abrogates the binding of the a2 integrin subunit I domain to collagen, provides unequi-vocal evidence that this leech product is a potent a2b1 blocker As the binding sites for VWF and a2b1on colla-gen are within close spatial proximity [26], saratin pre-sumably binds to an overlapping epitope on collagen to achieve dual blockade of these interactions Therefore,
D B
Fig 3 The effect of saratin on platelet adhesion on immobilized soluble collagen Human washed platelets (2 · 10 7 mL)1) were placed on soluble collagen-coated coverslips for 45 min at 37 C, and imaged using DIC microscopy In selected experi-ments, the function-blocking the a 2 b 1 mAb 6F1 (10 lgÆmL)1) or saratin (10 lgÆmL)1) was added to the platelet suspension either
in the absence (A, B) or in the presence (C, D) of thrombin (1 UÆmL)1) Experiments were performed in the absence (black bars)
or the presence (white bars) of the ADP-removing enzyme apyrase (apy) and the cy-clooxygenase inhibitor indomethacin (indo)
as indicated The numbers of adherent platelets were recorded for five fields of view (0.013 mm 2 ) and expressed as mean ± SEM from at least three experi-ments *P < 0.01 with respect to platelet adhesion in the absence of apy ⁄ indo for each respective treatment; **,dP < 0.01 with respect to platelet adhesion in the absence of receptor inhibitors and absence
or presence of apy ⁄ indo, respectively.
Trang 7in light of the role that a2b1plays in stabilizing
collagen-bound platelets under shear [5,6], the combined ability
of saratin to block both dependent and
VWF-independent (via a2b1) pathways of platelet deposition
on collagen makes this leech product a powerful anti-thrombotic agent
Experimental procedures
Reagents Fibrillar type I collagen (Horm) from equine tendon was purchased from Nycomed (Munich, Germany) Soluble, nonfibrillar type I collagen from rat tail was purchased from Sigma (St Loius, MO, USA) LJ-CP8 was generously provided by Z M Ruggeri (Scripps Research Institute,
La Jolla, CA, USA) 6F1 and 6D1 were a kind gift from
A
B
Fig 4 Saratin blocks Src kinase-independent platelet adhesion
on immobilized soluble collagen Human washed platelets
(2 · 10 7
mL)1) were placed on soluble collagen-coated coverslips for
45 min at 37 C and (A) imaged using DIC microscopy In selected
experiments, the function-blocking a2b1mAb 6F1 (10 lgÆmL)1) or
saratin (10 lgÆmL)1) was added to the platelet suspension either in
the absence or presence of the Src kinase inhibitor PP2 (20 l M ).
(B) The numbers of adherent platelets were recorded for five fields
of view (0.013 mm 2 ) and expressed as mean ± SEM from at least
three experiments *, dP < 0.01 with respect to platelet adhesion in
the absence or presence of PP2 for each respective inhibitor.
Fig 5 Saratin inhibits PRP adhesion on collagen Human platelets
in PRP were layered onto either a soluble (A) or fibrillar (B) collagen-coated slide for 45 min at 37 C and imaged using DIC microscopy In selected experiments, function-blocking a2b 1 mAb 6F1 (10 lgÆmL)1), GPIb mAb 6D1 (10 lgÆmL)1), aIIbb3mAb LJ-CP8 (CP8; 100 lgÆmL)1)
or saratin (10 lgÆmL)1) was added to PRP Images are representative
of at least three experiments.
Trang 8B Coller (Rockefeller University, New York, NY, USA).
The Src kinase inhibitor PP2 was purchased from
Calbio-chem (San Diego, CA, USA) Recombinant saratin,
pro-duced in the yeast Han polymorpha as previously described
[8], was supplied by BioVascular, Inc (La Jolla, CA, USA)
Other reagents were obtained from Sigma or previously
named sources [27,28]
Preparation of washed platelets
Human venous blood was drawn by venipuncture from
healthy volunteers into sodium citrate and acid⁄ citrate ⁄
dextrose as previously described [3] PRP was prepared by
centrifugation of whole blood at 200 g for 20 min (5702 R
centrifuge, Eppendorf, Hamburg, Germany, rotor
F-35-30-17) The platelets were then isolated from PRP by
centrifu-gation at 1000 g for 10 min (5702 R centrifuge, Eppendorf,
Hamburg, Germany, rotor F-35-30-17) in the presence of
prostacyclin (0.1 lgÆmL)1) The pellet was resuspended in
modified Hepes⁄ Tyrodes buffer (129 mm NaCl, 0.34 mm
Na2HPO4, 2.9 mm KCl, 12 mm NaHCO3, 20 mm Hepes,
5 mm glucose, 1 mm MgCl2, pH 7.3) containing
0.1 lgÆmL)1 prostacyclin, washed, and resuspended
(2· 107
mL)1) in Hepes⁄ Tyrode buffer
In selected experiments, platelet suspensions were treated
with 10 lgÆmL)1 6F1, 100 lgÆmL)1 LJ-CP8, 10 lgÆmL)1
6D1, 10 lgÆmL)1 saratin, 1 UÆmL)1 thrombin, 20 lm PP2,
and⁄ or 2 UÆmL)1 apyrase and 10 lm indomethacin for
10 min before use in the assays It is noteworthy that this dose of saratin is well above the IC50 reported for platelet– collagen binding [8] All experiments were performed in the absence of exogenously added Ca2+
Platelet adhesion assays Glass coverslips were incubated with a suspension of fibrillar collagen (100 lgÆmL)1) or soluble collagen (50 lgÆmL)1) overnight at 4C Surfaces were then blocked with denatured BSA (5 mgÆmL)1) for 1 h at room temperature, and this was followed by subsequent washing with NaCl⁄ Pi before use in spreading assays In selected experiments, collagen-coated surfaces were treated for 10 min with saratin (10 lgÆmL)1), and this was fol-lowed by washing with NaCl⁄ Pi Quiescent platelets failed
to bind or spread on surfaces coated with denatured BSA (Table 1)
For spreading experiments, washed platelets (2·
107mL)1) were incubated on collagen-coated coverslips at
37C for 45 min Subsequently, coverslips were gently washed with Hepes⁄ Tyrode buffer to remove unbound cells
Fig 6 Inhibition of a 2 I-bio binding on collagen by saratin Coverslips
coated with fibrillar soluble collagen were preincubated with either
vehicle or saratin (10 lgÆmL)1) for 10 min A constant amount of
0.3 l M a 2 I-bio was added, and bound a 2 I-bio was detected by
add-ing streptavidin–FITC and visualized usadd-ing DIC and fluorescence
microscopy Images are representative of three experiments.
Fig 7 Saratin inhibits platelet adhesion on immobilized collagen under flow Anticoagulated human whole blood was perfused over
a fibrillar collagen coverslip at a shear rate of 1000 s)1for 4 min In selected experiments, blood was pretreated for 10 min with 6D1 (10 lgÆmL)1) with or without 6F1 (10 lgÆmL)1) In separate experi-ments, collagen-coated coverslips were pretreated with saratin (10 lgÆmL)1) for 10 min, whereas saratin (30 lgÆmL)1) was main-tained in whole blood during flow Images are representative of at least three experiments.
Trang 9Platelet spreading was imaged using Ko¨hler illuminated
Nomarski DIC optics with a Zeiss 63· oil immersion
1.40 NA plan-apochromat lens on a Zeiss Axiovert 200M
microscope (Carl Zeiss, Thornwood, NY, USA), and
recor-ded using stallion 4.0 (Intelligent Imaging Innovations,
Inc., Denver, CO, USA) To compute the degree of
adhe-sion and surface area of spreading platelets, images were
manually outlined and quantified by determining the
num-ber of pixels within each outline using a Java plug-in for
image j software, as previously described [28] Imaging a
graticule under the same conditions allowed the conversion
of pixel size to micrometers
Flow adhesion studies
Glass coverslips were coated with fibrillar collagen as
des-cribed above Coverslips were assembled onto a flow
cham-ber (Glyotech, Gaithersburg, MD, USA) and mounted on
the stage of an inverted microscope (Zeiss Axiovert 200M)
In selected experiments, coverslips were treated with
10 lgÆmL)1 saratin for 10 min prior to the flow assay
PPACK (40 lm) anticoagulated whole blood was perfused
through the chamber for 3 min at a wall shear rate of
1000 s)1, and this was followed by washing for 4 min at the
same shear rate with modified Tyrodes buffer and imaged
using DIC microscopy
Measurement of platelet aggregation
To prepare heparinized PRP, blood was collected from
healthy human donors into syringes containing heparin
sodium (10 UÆmL)1final concentration) PRP was obtained
by centrifugation of heparinized blood at 200 g for 15 min
(5702 R centrifuge, Eppendorf, rotor F-35-30-17) Optical
aggregation studies were carried out using a Born
aggreg-ometer (Chronolog, Havertown, PA, USA) with high-speed
stirring (1200 r.p.m.) at 37C Platelet shape change and aggregation were monitored by measuring changes in light transmission as previously described [29]
Binding competition assays The recombinant a2I domain-encoding region was gener-ated, purified and biotinylated as previously described [30] Purified material was characterized by SDS⁄ PAGE, and the concentration of a2I-bio was quantified using a detergent compatible-protein assay (Biorad, Hercules, CA, USA)
Coverslips were coated overnight at 4C with 1 mgÆmL)1 fibrillar collagen Wells were then blocked with denatured BSA (5 mgÆmL)1) for 1 h at room temperature, and this was followed by subsequent washing with NaCl⁄ Pi before incubation with vehicle or saratin (10 lgÆmL)1) for 10 min
A constant amount of a2I-bio (0.3 lm) or VWF (10 lgÆmL)1) was then added and allowed to bind for
90 min Following copious washing, bound a2I-bio or VWF was detected by adding streptavidin–FITC or anti-VWF–FITC, respectively, for 1 h at RT, and visualized using fluorescence microscopy
Analysis of data Experiments were carried out at least three times, and ima-ges shown are representative data from one experiment Where applicable, results are shown as mean ± SEM The statistical significance of differences between means was determined by ANOVA If means were shown to be signifi-cantly different, multiple comparisons were preformed by the Tukey test Probability values of P < 0.01 were consid-ered to be statistically significant
Acknowledgements
We would like to thank Steve P Watson and Andras Gruber for stimulating discussions, and Dr Barry Col-ler for the generous gifts of 6F1 and 6D1 Tara C White and Michelle A Berny are ARCS scholars, and David K Robinson is the recipient of a Johnson scholarship Owen J T McCarty is supported by an American Heart Association Beginning Grant-in-Aid (0665512Z)
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