PLATELET INTERACTIONS WITH LIPOSOMES CARRYING RECOMBINANT PLATELET MEMBRANE GLYCOPROTEINS OR FIBRINOGEN: APPROACH TO PLATELET SUBSTITUTES

Abstract

Liposomes carrying both recombinant platelet membrane glycoproteins GPIa/IIa (rGPIa/IIa) and GPIbα (rGPIbα) (rGPIa/IIa-Ibα-liposomes), or fibrinogen (Fbg-liposomes) were prepared. Their interactions with platelets on a collagen surface under flow conditions were evaluated using a recirculating flow chamber, mounted on an epifluorescence microscope, which allows for real-time visualization of fluorescence-labeled liposomes or platelets interacting with the surface. Adhesion of platelets to the collagen surface increased with increasing the shear rate from 600 to 2400 s⁻¹. Also, the percentages of surface coverage of rGPIa/IIa-Ibα-liposomes or Fbg-liposomes increased with increasing platelet adhesion. These phenomena were attenuated by a peptide containing arginine-glycine-aspartic acid (RGD-peptide), or prostaglandin E₁ (PGE), but not by a peptide containing arginine-glycine-glutamic acid (RGE-peptide). In a homogeneous solution, rGPIa/IIa-Ibα liposomes and Fbg-liposomes enhanced platelet aggregation in a dose-dependent manner, as evaluated using an aggregometer. These findings suggest that rGPIa/IIa-Ibα-liposomes and Fbg-liposomes form aggregates at the site of injury in blood vessels, resulting in stationary adhesion together with activated platelets.

INTRODUCTION

Platelets have diverse functions and it will be a formidable task to develop platelet substitutes that have the entire set of platelet functions. The basic and important platelet functions for primary hemostasis are adhesion and aggregation. Initial platelet adhesion depends on the interaction of platelet membrane glyco-protein GPIb/IX/V complexes on platelets with von Willebrand factor (vWf) adsorbed on the collagen surface, which serves to tether platelets to the collagen surface [1-4]. The collagen receptors of the tethered platelets such as GPIa/IIa or GPVI then strongly bind to the collagen surface, activating platelets to form aggregates [5-8]. Therefore, reconstitution of the platelet functions, with a focus on adhesion and aggregation at the bleeding sites to plug holes in blood vessels, and to facilitate the function of the remaining platelets is a reasonable starting point in developing platelet substitutes.

The simplest type of artificial platelets might be particles carrying platelet membrane glycoproteins and/or ligands of the proteins involved in platelet adhesion and aggregation. Thus, liposomes carrying recombinant GPIa/IIa (rGPIa/IIa)and/or GPIbα (rGPIbα) were prepared, and their adhesive properties to the collagen or vWf surface were evaluated under flow conditions [9-12]. We previously reported that liposomes carrying rGPIbα (rGPIbα-liposomes) reversibly interact with the vWf surface under flow conditions, depending on the shear rate and the densities of the receptors and matrix, and that the interaction is directly related to shear rate [[10]]. Direct interaction of rGPIa/IIa with the collagen surface dominates the adhesion of liposomes carrying both rGPIa/IIa and rGPIbα (rGPIa/IIa-Ibα-liposomes) to the collagen surface at a low shear rate. At a high shear rate, tethering of rGPIa/IIa-Ibα-liposomes via the interaction between rGPIbα and the vWf-adsorbed collagen surface reduces the velocity of liposomes, enabling binding of rGPIa/IIa to the collagen surface [[9]], [11-12].

In the present study, we showed that rGPIa/IIa-Ibα-liposomes, or liposomes carrying fibrinogen (Fbg-liposomes) interact with activated platelets to enhance the formation of platelet aggregates.

MATERIALS AND METHODS

The RGD and RGE-peptides, PGE, and mepacrine were purchased from Sigma Chemical Co. (St. Louis, MO). Fibrinogen was obtained from Calbiochem. Co. (La Jolla, CA). Liposomes were prepared and characterized as described previously [[10]], [[12]]. Platelets and liposomes were fluorescence-labeled with mepacrine and rhodamine, respectively. The interaction of liposomes with platelets on the collagen surface was studied using a recirculating chamber mounted on an epifluorescence microscope as described previously [[10]], [[12]], with excitation and emission wavelengths of 550 and 590 nm for rhodamine, and 420 and 480 nm for mepacrine. An aggregometer, PA-100 (Kowa, Nagoya, Japan), was utilized to assess platelet aggregation in the presence or absence of liposomes.

RESULTS

Interaction of rGPIa/IIa-Ibα-Liposomes with Platelets on the Collagen Surface

Each single frame shown in Fig. 1A was obtained after a 3-min perfusion of a mixture of rGPIa/IIa-Ibα-liposomes and platelets on the collagen surface at a different shear rate, as indicated. When exposed to a shear rate of 600 S⁻¹ for 3min, the percentages of surface coverage of rGPIa/IIa-Ibα-liposomes and platelets were 5.41 ± 0.45% and 7.01 ± 1.51%, respectively (Fig. 1B). At a shear rate of 2400 S⁻¹, the surface coverage of rGPIa/IIa-Ibα-liposomes increased to 28.57 ± 2.13%, simultaneously with an increase in the surface coverage of platelets to 22.85 ± 2.06% (Fig. 1B).

Figure 1. Interaction of rGPIa/IIa-Ibα-liposomes with platelets on the collagen surface depends on the shear rate. Images were obtained after a 3-min perfusion of a mixture of liposomes (4.0 × 10⁵/μl) and platelets (1.0 × 10⁵/μl) on the surface at a hematocrit of 37.5%, soluble vWf concentration of 10 μg/ml, 2 mM Mg²⁺, 37°C, and various shear rates as indicated. Exofacial concentration of rGPIa/IIa and rGPIbα was 1.0 and 0.70 μg/ml, respectively. Values are the mean ± standard deviation, n = 6.

The adhesion and aggregation of rGPIa/IIa-Ibα-liposomes onto the collagen surface was abolished by the RGD-peptide or PGE, but not by the RGE-peptide. The effects of the RGD-peptide, RGE-peptide, and PGE are shown as a change in the relative surface coverage, i.e., the surface coverage in the presence of the peptide or PGE relative to that in the absence of the peptide or PGE. In the presence of 1 mM RGD-peptide, the relative surface coverage decreased from 90.3 ± 3.3% to 29.7 ± 1.1% when the shear rate was increased from 600 to 2400 S⁻¹ (Fig. 2). On the other hand, the RGE-peptide had no effect on the relative surface coverage of rGPIa/IIa-Ibα-liposomes (Fig. 2). The presence of 1 μM PGE reduced the relative surface coverage from 93.0 ± 4.1% to 46.6 ± 2.2% when the shear rate was increased from 600 to 2400 ⁻¹ (Fig. 2).

Figure 2. Inhibition of the interaction of rGPIa/IIa-Ibα-liposomes with platelets on the collagen surface. Experimental conditions were the same as described for Figure 1. Inhibition studies were performed in the presence of the RGD-peptide (1 mM), RGE-peptide (1 mM), or PGE (1 μM).

Enhancement of Platelet Aggregation by rGPIa/IIa-Ibα-Liposomes

Platelet aggregation is usually impaired when the platelet concentration is low. We examined the effect of rGPIa/IIa-Ibα-liposomes and control liposomes on platelet aggregation at a low platelet concentration using an aggregometer. Platelet rich plasma (PRP) at 1 × 10⁴/μl platelets was first mixed with various concentrations of rGPIa/IIa-Ibα-liposomes, and the mixture was then added to 10 μg/ml fibrous collagen. rGPIa/IIa-Ibα-liposomes dose-dependently enhanced platelet aggregation (Fig. 3). The effect of control liposomes at the same concentration of egg phosphatidylcholine (EPC) on platelet aggregation was negligible, suggesting that the enhancement was caused by specific interactions between platelets and rGPIa/IIa-Ibα-liposomes (Fig. 3).

Figure 3. Enhancement of platelet aggregation by rGPIa/IIa-Ibα-liposomes assessed by changes in light scattering intensity. PRP at 1 × 10⁴/μl platelets was mixed with various concentrations of rGPIa/IIa-Ibα-liposomes, and 10 μg/ml of collagen was then added to the mixture.

Interaction of Fbg-Liposomes with Platelets on the Collagen Surface

Each single frame shown in Fig. 4A was obtained after a 3-min perfusion of a mixture of Fbg-liposomes and platelets on the collagen surface at a different shear rate, as indicated. The percentages of surface coverage of Fbg-liposomes increased from 8.61 ± 0.79% to 19.87 ± 1.76% simultaneously with an increase in the surface coverage of platelets when the shear rate was increased from 600 to 2400 S⁻¹ (Fig. 4B).

Figure 4. Interaction of Fbg-liposomes with platelets on the collagen surface depends on the shear rate. Experimental conditions were the same as described for Figure 1, except that the exofacial concentration of Fbg was 0.74 μg/ml.

The adhesion and aggregation of Fbg-liposomes onto the collagen surface was attenuated by the RGD-peptide or PGE, but not by the RGE-peptide. The relative surface coverage of Fbg-liposomes decreased from 67.9 ± 2.9% to 10.7 ± 0.9%, and from 74.3 ± 3.6% to 20.1 ± 1.6%, when the shear rate was increased from 600 to 2400 S⁻¹, in the presence of 1 mM RGD-peptide and 1 μM PGE, respectively (Fig. 5). The RGE-peptide had no effect on the relative surface coverage of Fbg-liposomes (Fig. 5).

Figure 5. Inhibition of the interaction of Fbg-liposomes with platelets on the collagen surface. Experimental conditions were the same as described for Figure 2.

Enhancement of Platelet Aggregation by Fbg-Liposomes

PRP at 5 × 10⁴/μl platelets was first mixed with various concentrations of Fbg-liposomes, and 2 μM ADP was then added to the mixture. Fbg-liposomes dose-dependently enhanced platelet aggregation (Fig. 6). Control liposomes at the same concentration of EPC enhanced the formation of small aggregates, however, the small aggregates failed to convert to medium or large aggregates (Fig. 6).

Figure 6. Enhancement of platelet aggregation by Fbg-liposomes assessed by changes in light scattering intensity. PRP at 5 × 10⁴/μl platelets was mixed with various concentrations of Fbg-liposomes, and 2 μM ADP was then added to the mixture.

DISCUSSION

The interaction of rGPIa/IIa-Ibα-liposomes with platelets onto the collagen surface was directly related to shear rate, and was inhibited by the RGD-peptide, which interferes with the interaction of GPIIb/IIIa with the RGDS sequence of vWf located at residues 1744 to 1747 in the carboxyl terminal C1 domain [[13]]. The interaction was also abolished by PGE, which activates adenylate cyclase, and thereby increases the cytoplasmic cyclic AMP level, decreases the intracellular calcium concentration, and suppresses the signal transduction, activation of GPIIb/IIIa, and the release of thromboxane A₂ or serotonin [[14]]. These results suggest that rGPIa/IIa-Ibα-liposomes form the aggregates through an interaction between rGPIbα on the liposome surface with activated GPIIb/IIIa via vWf, especially at high shear rates. Alternatively, rGPIbα on the liposome surface might interact with vWf expressed on activated platelets, thereby forming aggregates.

The interaction of Fbg-liposomes with platelets onto the collagen surface was also directly related to shear rate, and was abolished by the RGD-peptide or PGE, but not by the RGE-peptide, indicating that Fbg-liposomes bind to activated platelets through an interaction of the AGDV sequence on fibrinogen located at the γ-chain with activated GPIIb/IIIa [[15]].

In conclusion, we showed that rGPIa/IIa-Ibα-liposomes and Fbg-liposomes might interact with activated platelets to form aggregates at the site of injury in blood vessels, although their mechanisms are different.

ACKNOWLEDGMENTS

This work was supported by Health Science Research Grants, Research on Advanced Medical Technology, Ministry of Health and Welfare, Japan. We thank the Welfide Corporation (Osaka, Japan) for preparation of rGPIbα and vWf.