Thrombin Mutant W215A/E217A Acts as a Platelet GPIb Antagonist

Reagents

The recombinant human thrombin mutants Trp215Ala/Glu217Ala (WE), Thr172Tyr/Glu217Ala (TW), Thr172Tyr/Trp215Ala/Glu217Ala (TWE), and Ser195Ala (S195A) were expressed, purified, and tested as previously described.^6,10–12 Thrombin mutants were concentrated to 10 mg/mL in 50 mmol/L choline chloride, 20 mmol/L 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0. It is noteworthy that presence of diluent (1:500; corresponding to 20 μg/mL thrombin mutant) alone did not affect platelet function in any of the assays tested. vWF was a kind gift from Dr Michael Berndt (Monash University, Clayton, Australia). The anti-GPIb monoclonal antibody (mAb) 6D1 was a generous gift from Dr Barry Coller (Rockefeller University, New York), and the anti-GPIb mAb SZ2 was purchased from Beckman Coulter (Fullerton, Calif). Prothrombin and fibrinogen were purchased from Enzyme Research Laboratories, Inc. Chinese hamster ovary (CHO) cells stably expressing the platelet glycoprotein (GP) Ib-IX complex (CHOαβIX cells) or only GP Ibβ and IX (CHOβIX cells)¹³ were kind gifts from Dr José López (Puget Sound Blood Center, Seattle, Wash). Other reagents were from Sigma-Aldrich Inc or previously named sources.^14,15

Preparation of Human and Murine Washed Platelets

Human venous blood was drawn by venipuncture from healthy volunteers into sodium citrate and acid/citrate/dextrose (ACD) as previously described.^14,15 The generation of mice deficient in either Rac1 or WAVE-1 was as previously described,^14,16 and were kindly donated by Drs Laura Machesky and Steve Watson (University of Birmingham, UK) and Victor Tybulewicz (National Institute for Medical Research, London, UK). After centrifugation steps, purified human or murine platelets were resuspended in modified Tyrodes buffer (129 NaCl mM, 0.34 mmol/L Na₂HPO₄, 2.9 mmol/L KCl, 12 mmol/L NaHCO₃, 20 mmol/L HEPES, 5 mmol/L glucose, 1 mmol/L MgCl₂; pH 7.3). Autologous human red blood cells (RBC) were isolated as previously described.^14,15 Washed platelets were then reconstituted with 50% (v/v) autologous packed RBCs (reconstituted blood). All animals were maintained using housing and husbandry in accordance with local and national legal regulations, and human donors provided informed written consent, according to the Declaration of Helsinki.

Platelet Static and Flow Adhesion Assays

Glass coverslips were incubated with a solution of wild-type or mutant thrombin (50 μg/mL), prothrombin (50 μg/mL), collagen (100 μg/mL), fibrinogen (50 μg/mL), or vWF (50 μg/mL) overnight at 4°C as described.^14,15 Surfaces were then blocked with denatured bovine serum albumin (BSA, 5 mg/mL) for 1 hour followed by washing with phosphate-buffered saline (PBS) before use in adhesion assays.

For static spreading experiments, washed platelets (2×10⁷/mL) were incubated on protein-coated coverslips at 37°C for 45 minutes, followed by washing with modified Tyrodes buffer to remove unbound cells.^14,15 Platelet spreading was imaged using Köhler illuminated Nomarski differential interference contrast (DIC) optics with a Zeiss 63× oil immersion 1.40 NA plan-apochromat lens on a Zeiss Axiovert 200 mol/L microscope (Carl Zeiss) and recorded using Stallion 4.0 (Intelligent Imaging Innovations Inc). The degree of adhesion and surface area coverage was computed using Image J software as previously described.^14,15

For flow adhesion assays, protein-coated coverslips were assembled onto a flow chamber (Glyotech) and mounted on the stage of an inverted microscope (Zeiss Axiovert 200M). In selected experiments, reconstituted blood or PPACK (40 μmol/L) anticoagulated whole blood was perfused through the chamber for 3 minutes at a wall shear rate of 300 to 1000 s⁻¹, and imaged using DIC microscopy in both real-time and after washing with modified Tyrodes buffer. The number of tethering and rolling platelets during the assay was determined off-line by reviewing the real-time videos as previously described.^14,15

Analysis of Data

Statistical significance of differences between means was determined by ANOVA. If means were shown to be significantly different, multiple comparisons were performed by the Tukey test.^14,15 Probability values of P<0.01 were selected to be statistically significant.

Molecular Mechanisms of Platelet Cytoskeletal Reorganization on Thrombin-Coated Surfaces

To characterize the molecular basis of platelet–thrombin interactions, we gently pipetted purified platelets over surface immobilized thrombin and monitored platelet morphology using Nomarski DIC microscopy. To assess the direct ability of thrombin to support platelet binding, these experiments were performed in the presence of apyrase and indomethacin, inhibitors of the secondary mediators ADP and thromboxane A₂ (TxA₂), respectively. Consistent with our previous reports,¹⁷ immobilized thrombin supported human platelet adhesion and lamellipodia formation (Figure 1). In light of the fact that thrombin has been shown to induce robust activation of the small GTPase Rac1 in platelets,¹⁴ we aimed to investigate the role of Rac1 in mediating platelet spreading on immobilized thrombin. As shown in Figure 1, immobilized thrombin supported wild-type murine platelet adhesion and activation, as evidenced by generation of filopodia and full lamellipodia. Strikingly, lamellipodia formation was abolished in Rac1-deficient platelets on thrombin, although Rac1^−/− platelets retained the ability to form filopodia. In contrast, full lamellipodia formation was observed in the absence of the actin scaffolding protein WAVE-1 (Figure 1). The presence of the actin polymerization-inhibitor cytochalasin D abrogated both filopodia and lamellipodia formation for wild-type, Rac1^−/−, and WAVE-1^−/− platelets on thrombin.

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Figure 1. The role of Rac1 and WAVE-1 on spreading of platelets on immobilized thrombin. Washed human platelets (2×10⁷/mL) or murine platelets (2×10⁷/mL) from wild-type, Rac1^−/−, or WAVE-1^−/− mice were placed on thrombin coated coverslips for 45 minutes at 37°C and imaged using DIC microscopy. Experiments were performed in the absence or presence of the actin-polymerization inhibitor cytochalasin D (Cyto D; 10 μmol/L). Images are representative of at least 3 experiments.

We next investigated the role of proteolysis in platelet adhesion to immobilized thrombin. Our results demonstrate that platelet adhesion and activation were abrogated in the presence of PPACK (40 μmol/L), which blocks the active site of thrombin (Figure 2). Similarly, platelets failed to adhere to surface-immobilized thrombin Na⁺-binding site mutants WE, TW, or TWE (Figure 2). Platelets failed to bind to the active site mutant S195A, which has been shown to retain the Na⁺-binding properties of wild-type thrombin¹² or to surface-immobilized prothrombin or BSA (Figure 2). It is noteworthy that the presence of WE in solution did not affect the degree of platelet adhesion to immobilized thrombin, collagen or fibrinogen under static conditions (supplemental Table I, available online at http://atvb.ahajournals.org). Moreover, both the rate and extent of thrombin-induced platelet aggregation was unaffected by the presence of WE (data not shown).

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Figure 2. Platelet binding to wild-type or mutant thrombin. Washed human platelets (2×10⁷/mL) were placed on coverslips coated with either thrombin (Thr), PPACK-treated thrombin, WE, TW, TWE, S195A, prothrombin (PT), or BSA for 45 minutes at 37°C, and imaged using DIC microscopy. The number of adherent platelets were recorded for 3 fields of view (0.013 mm²) and expressed as mean±SEM from at least 3 experiments.

Taken together, our data show that Rac1, but not WAVE-1, is required for regulating platelet lamellipodia formation on thrombin, and that this mechanism is not activated by immobilized WE.

Platelet Interactions With Immobilized Thrombin Under Flow

We investigated thrombin-platelet interactions under shear stress that better characterized the microenvironment of thrombus formation in the intraluminal boundary layer of blood flow. We assessed platelet adhesion and aggregation after perfusion of reconstituted blood over immobilized thrombin at a shear rate of 300 s⁻¹. Tightly-packed platelet aggregates (33.5±1.3 aggregates/mm² × 10⁻²) consisting of 10 to 50 platelets were seen on thrombin after perfusion of reconstituted blood for 3 minutes (Figure 3A). It is noteworthy that these platelet aggregates were stable and that we did not observe a significant level of embolization. In accord with previous reports,¹⁸ platelet aggregation on thrombin was significantly reduced in the presence of the blocking anti-GPIb mAb SZ2 (20 μg/mL), resulting in 46.4±1.3 aggregates/mm² × 10⁻² consisting of 1 to 5 platelets (Figure 3B). Interestingly, platelet aggregation on immobilized thrombin in the presence of exogenously added WE (20 μg/mL) was reduced to a similar degree as was observed in the presence of SZ2, resulting in 45.9±1.4 aggregates/mm² × 10⁻² consisting of 1 to 5 platelets (Figure 3C). This was further evidenced by a reduction in the percentage of platelet aggregate surface coverage on thrombin in the presence of WE (58.5±4.2% versus 16.8±3.6% surface coverage on thrombin in the presence of vehicle or WE, respectively). Minimal platelet adhesion was observed on thrombin in the presence of PPACK (Figure 3D), whereas platelets failed to firmly adhere on WE, S195A, prothrombin, or BSA under flow (Figure 3E through 3H).

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Figure 3. Platelet adhesion to wild-type or mutant thrombin under flow. Reconstituted human blood was perfused at a shear rate of 300s⁻¹ for 3 minutes over thrombin-coated slides in the presence of vehicle (A), the blocking GPIb mAb SZ2 (B), exogenously added WE (C), or PPACK (D). In separate experiments, reconstituted blood was perfused over immobilized WE (E), S195A (F), prothrombin (G), or BSA (H). Images are representative of at least 3 experiments.

As activation of platelets is not mandatory for GPIb-mediated binding to ligands such as vWF,¹⁹ we aimed to investigate whether inactive thrombin, which failed to support firm adhesion (Figure 3D), retained the ability of support GPIb-mediated events. We perfused PPACK-anticoagulated whole blood over immobilized thrombin and monitored platelet interactions under shear flow conditions in real-time using Nomarski DIC microscopy. Platelet tethering and rolling was observed on immobilized wild-type thrombin in whole blood (Figure 4A and 4B). Furthermore, platelet tethering and rolling was observed on WE thrombin, although to a lesser extent than compared with wild-type thrombin (Figure 4C). Platelets failed to arrest and adhere on wild-type or WE thrombin in the presence of PPACK, suggesting that a proteolytic event was required for platelet firm adhesion. The presence of either the function-blocking anti-GPIb mAb SZ2 or exogenously added WE (20 μg/mL) in solution abrogated platelet interactions on both immobilized wild-type and WE thrombin (Figure 4). In contrast, the presence of vehicle, wild-type thrombin, or prothrombin (20 μg/mL) in solution did not affect the extent of platelet tethering and rolling. Platelets failed to interact with immobilized prothrombin or BSA (data not shown).

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Figure 4. The platelet receptor GPIb binds to thrombin and WE. Human whole blood anticoagulated with PPACK was perfused over wild-type thrombin-coated or WE-coated slides at a shear rate of 300 s⁻¹ for 3 minutes. A, Representative micrographs of platelet tethering on wild-type thrombin in the presence of vehicle, the blocking GPIb mAb SZ2, or WE (20 μg/mL) are shown. The cumulative number of tethering platelets on wild-type thrombin (B) or WE (C) was recorded for a single field of view (0.013 mm²) for 30 seconds and expressed as mean±SEM from at least 3 experiments. *P<0.01 with respect to vehicle.

To assess the ability of WE to bind to GPIbα, radiolabeled WE thrombin (¹²⁵I-WE) was incubated with CHOαβIX cells expressing GPIbα (and GPIbβ and GPIX to facilitate GPIbα expression). Our data show that GPIbα-expressing CHO cells bound 2.1-fold more ¹²⁵I-WE as compared with CHOβIX cells, which lack GPIbα (supplemental Figure I). Moreover, ¹²⁵I-WE binding to CHOαβIX cells was reduced by nearly 50% in the presence of the anti-GPIb mAb SZ2. CHOαβIX cells supported 6.4-fold more binding of ¹²⁵I-WE relative to ¹²⁵I-BSA. Additionally, CHOαβIX cells were observed to tether and roll when perfused over immobilized WE at a shear rate of 150 s⁻¹, and this rolling was inhibited by the presence of the anti-GPIb mAb SZ2 (data not shown). Overall, our data demonstrate that WE, although unable to support PAR-dependent platelet activation, retains the ability to bind GPIb, supports GPIb-dependent platelet rolling, and inhibits GPIb-dependent platelet tethering on thrombin under flow conditions that are typical of the intravascular environment.

The Thrombin Mutant WE Inhibits Platelet Adhesion to Collagen and Rolling on vWF Under Flow Conditions

It is well established that GPIb plays an essential role in mediating platelet aggregate formation on immobilized collagen under shear flow.^20,21 In accord with these reports, our data demonstrate that perfusion of PPACK-anticoagulated whole blood over collagen resulted in substantial platelet aggregate formation on collagen fibers at a shear rate of 1000 s⁻¹ (Figure 5A), whereas platelet adhesion and aggregation was severely reduced in the presence of the GPIb mAb 6D1 (Figure 5B). Strikingly, the presence of WE in solution also inhibited platelet deposition on collagen (Figure 5C) in a dose-dependent manner with an IC₅₀ of 7.98 μg/mL (supplemental Figure II). In contrast, presence of vehicle, wild-type thrombin (20 μg/mL), S195A (20 μg/mL), or prothrombin (20 μg/mL) did not reduce platelet aggregate formation on collagen (Figure 5D through 5F).

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Figure 5. The thrombin mutant WE inhibits platelet adhesion on immobilized collagen under flow. Human whole blood anticoagulated with PPACK was perfused over collagen-coated slides at a shear rate of 1000 s⁻¹ for 3 minutes. In selected experiments, whole blood was incubated with vehicle (A), the blocking GPIb mAb 6D1 (B), WE (C), wild-type thrombin (Thr) (D), S195A (E), or prothrombin (PT) (F) at a final concentration of 20 μg/mL. Images are representative of at least 3 experiments.

To determine the molecular mechanisms by which WE inhibits platelet aggregation on collagen under flow, we perfused PPACK-anticoagulated whole blood over immobilized vWF at a shear rate of 1000 s⁻¹ and monitored platelet tethering and rolling in real-time with DIC microscopy. Our results show that platelets tethered and rolled on vWF under flow (Figure 6A), while the presence of the anti-GPIb mAb 6D1 dramatically reduced the degree of rolling platelets (Figure 6B). Importantly, the presence of WE in solution dramatically reduced the degree of platelet rolling on vWF (Figure 6A and 6B). In contrast, the presence of vehicle, wild-type thrombin, S195A, or prothrombin had no effect on platelet-vWF interactions (Figure 6B). Taken together, our data suggest that WE acts as a potent platelet GPIb antagonist by blocking vWF-platelet binding under shear flow conditions that typically occur at the blood–thrombus interface, whereas it does not appear to block platelet adhesion and activation in the absence of shear, which typically occurs in wound blood.

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Figure 6. Evaluation of platelet rolling on immobilized vWF under flow. Human whole blood anticoagulated with PPACK was perfused over vWF-coated slides at a shear rate of 1000 s⁻¹. A, Representative micrographs of platelet tethering on vWF in the presence of vehicle or WE (20 μg/mL). B, In selected experiments, whole blood was incubated with either vehicle, the blocking GPIb mAb 6D1, WE, wild-type thrombin (Thr), S195A, or prothrombin (PT). The number of tethering platelets was recorded at a single time point for each field of view (0.013 mm²) after 30 seconds of flow and expressed as mean±SEM.