The leech product saratin is a potent inhibitor of platelet integrin a 2 b 1 and von Willebrand factor binding to collagen Tara C. White 1 , Michelle A. Berny 1 , David K. Robinson 1 , Hang Yin 2 , William F. DeGrado 2,3 , Stephen R. Hanson 1 and Owen J. T. McCarty 1,4 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 a 2 b 1 ; 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 a 2 b 1 to 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 A 2 , both saratin and 6F1, a blocking a 2 b 1 mAb, abro- gated platelet adhesion to fibrillar and soluble collagen. Additionally, sara- tin eliminated a 2 b 1 -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 a 2 b 1 and von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the a 2 integrin 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 a 2 b 1 . These results demonstrate that saratin interferes with inte- grin a 2 b 1 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; TxA 2 , thromboxane A 2 ; VWF, von Willebrand factor. FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS 1481 translocation at the site of injury, allowing adhesive interactions with slower binding kinetics (such as the platelet collagen receptors GPVI and a 2 b 1 and a IIb b 3 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 a 2 b 1 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 a 2 b 1 can 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 a 2 b 1 . 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 a 2 b 1 integrin, namely the homologous integrin a 2 I domain [26]. The present study demonstrates that saratin interferes with integrin a 2 b 1 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 a 2 b 1 - blocking antibody 6F1 (data not shown), consistent with previous reports demonstrating an a 2 b 1 -depend- ent lag phase for collagen-induced aggregation [29]. Together, these findings led us to question whether saratin blocks platelet a 2 b 1 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 a 2 b 1 -blocking antibody or saratin; however, these effects were statistically insigni- Saratin blocks a 2 b 1 -collagen binding T. C. White et al. 1482 FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS 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 A 2 (TxA 2 ), respectively (Fig. 2B). Furthermore, the integrin a 2 b 1 mediates this ADP ⁄ TxA 2 -independent platelet adhesion to fibrillar collagen, as evidenced by the abrogation of platelet binding in the presence of the a 2 b 1 mAb 6F1 (Fig. 2A). A long the se lines, ADP ⁄ TxA 2 - 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 a 2 b 1 integrins [31,32]. Indeed, the presence of the a 2 b 1 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 ⁄ TxA 2 - independent platelet adhesion to soluble collagen was eliminated through the blockade of a 2 b 1 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 a 2 b 1 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 a IIb b 3 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. T. C. White et al. Saratin blocks a 2 b 1 -collagen binding FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS 1483 itor 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 a 2 b 1 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 a 2 b 1 -mediated platelet bind- ing. It is noteworthy that this series of experiments was performed in the absence of inhibitors of ADP and TxA 2 . 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 a 2 b 1 mAb 6F1 or saratin abro- gated this adhesion (Fig. 5A), further demonstrating the ability of saratin to block a 2 b 1 -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 )2 platelets ⁄ mm 2 on soluble collagen in the presence or absence of 10 lgÆmL )1 sar- atin, respectively; mean ± SEM; n ¼ 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 a 2 b 1 mAb 6F1 was used in combination with A C B D 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 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 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; d P < 0.01 with respect to platelet adhesion in the presence of apy ⁄ indo and absence of 6D1 or saratin. Saratin blocks a 2 b 1 -collagen binding T. C. White et al. 1484 FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS antagonists to VWF receptors on platelets, namely the mAb to GPIb, 6D1, and the mAb to a IIb b 3 , LJ-CP8. Taken together, these data are reflective of the ability of saratin to both block VWF–collagen binding and to inhibit a 2 b 1 –collagen interactions. Inhibition of a 2 integrin subunit I domain binding to collagen by saratin In an attempt to determine whether the binding site on collagen for the platelet receptor a 2 b 1 is blocked by the leech product saratin, we utilized a biotinylated recombinant a 2 integrin subunit I domain construct. Previous studies have shown that the a 2 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 a 2 b 1 binding, coverslips were coated with fibrillar collagen type I and preincu- bated with or without saratin. Subsequently, a con- stant amount of biotinylated a 2 integrin subunit I domain (a 2 I-bio) was added, and the amount of a 2 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 a 2 I-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 a 2 b 1 binding to collagen in addi- tion to blocking VWF–collagen interactions. 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 a 2 b 1 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 a 2 b 1 antagonists. Similar results were observed in reconstituted blood (data not shown). Altogether, our results demonstrate that sara- tin, through blockade of both VWF and a 2 b 1 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 a 2 b 1 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 a 2 b 1 integrins are not Table 1. Effects of an a 2 b 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 lM), 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 fibrinogen was treated with the a 2 b 1 -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 ) BSA – – 2.2 ± 0.62* , ** Fibrillar collagen – – 43.7 ± 2.57 Fibrillar collagen 6F1 – 44.2 ± 1.98 Fibrillar collagen – 6F1 8.1 ± 0.25* Fibrillar collagen Saratin – 7.9 ± 1.45* Fibrillar collagen – Saratin 8.2 ± 0.80* Soluble collagen – – 30.1 ± 1.50 Soluble collagen 6F1 – 31.6 ± 1.21 Soluble collagen – 6F1 2.9 ± 0.74** Soluble collagen Saratin – 2.7 ± 0.59** Soluble collagen – Saratin 2.8 ± 0.80** Fibrinogen – – 63.2 ± 1.03 Fibrinogen Saratin – 62.8 ± 1.88 Fibrinogen – Saratin 64.4 ± 1.60 * , **P < 0.01 with respect to platelet adhesion ⁄ surface area on untreated fibrillar or soluble collagen, respectively. T. C. White et al. Saratin blocks a 2 b 1 -collagen binding FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS 1485 essential 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 TxA 2 ), which leads to platelet adhesion independently of a 2 b 1 . 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 a 2 b 1 , 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 signaling [1,35]. Consistent with previous reports [32–34,36,37], our data demonstrate that a 2 b 1 integrins play an important role in mediating platelet adhesion to soluble collagen in the absence of inhibi- tors of secondary mediators, whereas a 2 b 1 is essential for ADP ⁄ TxA 2 -independent platelet adhesion. Addi- tionally, we demonstrate that PRP binding to immobi- lized soluble collagen is a 2 b 1 -dependent. Moreover, the a 2 b 1 dependency of Src kinase-independent adhesion on soluble collagen further indicates the essential role of a 2 b 1 in the absence of platelet activation. Interest- ingly, we found that thrombin stimulation, which pre- dominantly acts via the G q family of proteins [38], potentiated a 2 b 1 -independent platelet adhesion to sol- uble collagen only in the presence of the actions of the G i 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 G i -coupled receptors in the absence of a 2 b 1 [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 a 2 integrin subunit I domain to collagen, provides unequi- vocal evidence that this leech product is a potent a 2 b 1 blocker. As the binding sites for VWF and a 2 b 1 on 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, A C DB 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. Saratin blocks a 2 b 1 -collagen binding T. C. White et al. 1486 FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS in light of the role that a 2 b 1 plays in stabilizing collagen- bound platelets under shear [5,6], the combined ability of saratin to block both VWF-dependent and VWF- independent (via a 2 b 1 ) 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 a 2 b 1 mAb 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. AB 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 a 2 b 1 mAb 6F1 (10 lgÆmL )1 ), GPIb mAb 6D1 (10 lgÆmL )1 ), a IIb b 3 mAb LJ-CP8 (CP8; 100 lgÆmL )1 ) or saratin (10 lgÆmL )1 ) was added to PRP. Images are representative of at least three experiments. T. C. White et al. Saratin blocks a 2 b 1 -collagen binding FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS 1487 B. 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 Na 2 HPO 4 ,2.9mm KCl, 12 mm NaHCO 3 ,20mm Hepes, 5mm glucose, 1 mm MgCl 2 , pH 7.3) containing 0.1 lgÆmL )1 prostacyclin, washed, and resuspended (2 · 10 7 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 Ca 2+ . Platelet adhesion assays Glass coverslips were incubated with a suspension of fibrillar collagen (100 lgÆmL )1 ) or soluble collagen (50 lgÆmL )1 ) overnight at 4 °C. 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 ⁄ P i 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 ⁄ P i . Quiescent platelets failed to bind or spread on surfaces coated with denatured BSA (Table 1). For spreading experiments, washed platelets (2 · 10 7 mL )1 ) were incubated on collagen-coated coverslips at 37 °C 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 using 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 )1 for 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. Saratin blocks a 2 b 1 -collagen binding T. C. White et al. 1488 FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS Platelet 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 )1 final 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 37 °C. Platelet shape change and aggregation were monitored by measuring changes in light transmission as previously described [29]. Binding competition assays The recombinant a 2 I domain-encoding region was gener- ated, purified and biotinylated as previously described [30]. Purified material was characterized by SDS ⁄ PAGE, and the concentration of a 2 I-bio was quantified using a detergent compatible-protein assay (Biorad, Hercules, CA, USA). Coverslips were coated overnight at 4 °C 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 ⁄ P i before incubation with vehicle or saratin (10 lgÆmL )1 ) for 10 min. A constant amount of a 2 I-bio (0.3 l m) or VWF (10 lgÆmL )1 ) was then added and allowed to bind for 90 min. Following copious washing, bound a 2 I-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). References 1 Nieswandt B & Watson SP (2003) Platelet–collagen interaction: is GPVI the central receptor? Blood 102, 449–461. 2 Ruggeri ZM (2006) Platelet interactions with vessel wall components during thrombogenesis. Blood Cells Mol Dis 36, 145–147. Table 2. Effects of saratin on platelet adhesion ⁄ aggregation on col- lagen under flow. Human whole blood was perfused over immobi- lized collagen at 1000 s )1 . Blood was pretreated for 10 min with the GPIb mAb 6D1 (10 lgÆmL )1 ) with or without the a 2 b 1 mAb 6F1 (10 lgÆmL )1 ). In separate experiments, collagen-coated coverslips were pretreated with saratin (10 lgÆmL )1 ) for 10 min, whereas sar- atin (30 lgÆmL )1 ) was maintained in whole blood during flow. Val- ues are reported as mean ± SEM of three experiments. Treatment Surface coverage (%) Number of thrombi ⁄ field of view – 34.7 ± 6.18 39.6 ± 1.95 6D1 6.9 ± 2.77* 36.4 ± 5.85 6D1 + 6F1 3.8 ± 0.96* 13.0 ± 4.19** Saratin 5.7 ± 2.43* 15.4 ± 5.27** * , **P < 0.01 with respect to untreated and 6D1-treated blood, respectively. T. C. White et al. Saratin blocks a 2 b 1 -collagen binding FEBS Journal 274 (2007) 1481–1491 ª 2007 The Authors Journal compilation ª 2007 FEBS 1489 3 McCarty OJ, Calaminus SDJ, Berndt MC, Machesky LM & Watson SP (2006) von Willebrand factor med- iates platelet spreading through glycoprotein Ib and a IIb b 3 in the presence of botrocetin and ristocetin, respectively. 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Robinson 1 , Hang. 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. von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the a 2 integrin subunit I domain to col- lagen, and prevented platelet adhesion to collagen