The recombinant fragment of the platelet membrane glycoprotein Ia/IIa (rGPIa/IIa) was conjugated to the polymerized albumin particles (polyAlb) with the average diameter of 180nm. The intravenous administration of rGPIa/IIa–polyAlb to thrombocytopenic mice ([platelet]=2.1±0.3×105particles/μL) with three doses of ca. 2.4×1010, 7.2×1010, and 2.4×1011particles/kg, respectively, significantly reduced their bleeding time to 426±71, 378±101, and 337±46s, respectively, whereas that of the control groups (PBS) was 730±198s. The injection of rGPIa/IIa–polyAlb (2.4×1011particles/kg) was approximately equal to the effect of the injection of the mouse platelets at a dose of 2.0×1010 particles/kg. It was confirmed that rGPIa/IIa–polyAlb had a recognition ability against collagen and could contribute to the hemostasis in the thrombocytopenic mice as a platelet substitute.

Integrin α2β1, which is a membrane protein consisting of noncovalently bound α2 and β1 chains, mediates cell binding to collagen and plays a role in platelet functions. DNAs encoding the chimeric proteins in which the extracellular domains of each α2 and β1 chain was fused to hinge and Fc regions of human IgG1γ chain were cotransfected into CHO cells. Soluble integrin α2β1 (sα2β1) in which α2 and β1 chains were covalently bound by disulfide bonds was recovered from the culture supernatant. sα2β1 maintained functional characteristics of cell surface α2β1 as indicated by cation-dependent binding to collagen and conformational changes induced by cations or ligand. Intravenously administered sα2β1 in rats colocalized with collagen in inflamed microvessels. Moreover, sα2β1-conjugated liposome administered intravenously reduced bleeding time of the thrombocytopenic mice. These results indicated that sα2β1 has pharmaceutical utilities as an agent for detecting injured vessels and a component of platelet substitute.

Liposomes carrying both recombinant glycoprotein Ia/IIa (rGPIa/IIa) and Ib alpha (rGPIb alpha) (rGPIa/IIa-Ib alpha-liposomes) instantaneously and irreversibly adhered to the collagen surface in the presence of soluble von Willebrand factor (VWF) at high shear rates, in marked contrast with translocation of liposomes carrying rGPIb alpha alone on the VWF surface. In the absence of soluble VWF, the adhesion of rGPIa/IIa-Ib alpha-liposomes to the collagen surface decreased with increasing shear rates, similar to liposomes carrying rGPIa/IIa alone. While adhesion of liposomes with exofacial rGPIa/IIa and rGPIb alpha densities of 2.17 x 10(3) and 1.00 x 10(4) molecules per particle, respectively, was efficient at high shear rates, reduction in rGPIb alpha density to 5.27 x 10(3) molecules per particle resulted in decreased adhesion even in the presence of soluble VWF. A 50% reduction in the exofacial rGPIa/IIa density resulted in a marked decrease in the adhesive ability of the liposomes at all shear rates tested. The inhibitory effect of antibody against GPIb alpha (GUR83-35) on liposome adhesion was greater at higher shear rates. Further, the anti-GPIa antibody (Gi9) inhibited liposome adhesion more than GUR83-35 at all shear rates tested. These results suggest that the rGPIa/IIa-collagen interaction dominates the adhesion of rGPIa/IIa-Ib alpha-liposomes to the collagen surface at low shear rates, while the rGPIa/IIa-collagen and rGPIb alpha-VWF interaction complements each other, and they synergistically provide the needed functional integration required for liposome adhesion at high shear rates. This study thus has confirmed for the first time the proposed mechanisms of platelet adhesion to the collagen surface under flow conditions using the liposome system.

Liposomes carrying both recombinant platelet membrane glycoproteins GPIa/IIa (rGPIa/IIa) and GPIb alpha (rGPIb alpha) (rGPIa/IIa-Ib alpha-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-Ib alpha-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-Ib alpha-liposomes and Fbg-liposomes enhanced platelet aggregation in a dose-dependent manner, as evaluated using an aggregometer. These findings suggest that rGPIa/IIa-Ib alpha-liposomes and Fbg-liposomes form aggregates at the site of injury in blood vessels, resulting in stationary adhesion together with activated platelets.

Effect of beraprost sodium (BPS), a long-acting and orally active stable analogue of PGI2, on the macromolecular permeability of cultured vascular endothelial cells (HUVEC) was detected by the transport of FITC-albumin. Thrombin treatment resulted in induction of FITC-albumin transport across the endothelial cell monolayer. The albumin transport induced by thrombin was not accompanied by any damage to the cells. BPS had no effect on the permeability of resting endothelial monolayers, while BPS inhibited the thrombin-induced increase in the albumin permeability in a dose-dependent manner (30-1000 nM). Treatment of the cells with PGI2 or dibutyryl cAMP caused a significant inhibition of the thrombin-induced increase in the albumin permeability. These results strongly suggested that BPS suppressed the thrombin-induced macromolecular permeability in HUVEC through the elevation of its intracellular cAMP, and that BPS was a suppressor against inflammatory vascular changes such as exudation.

Using a peroxide-injured endothelial cell model, beraprost sodium (beraprost) was tested in relation to the action to protect against the damage of vascular endothelial cells employing the viability of the cells and change in lipid peroxides as the indicators. At a concentration of 1-3 mumol/l or higher, beraprost significantly inhibited the decrease in viability of the cells and the increase in lipid peroxides level caused by t-butyl hydroperoxide or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. When similar tests were conducted with prostaglandin I2, its inhibiting action was equal to or slightly weaker than that of beraprost unlike PGE1 and PGD2. This action of beraprost in inhibiting cell damage caused by peroxides suggests that beraprost may be useful for protecting cells from damage due to ischemic diseases.

In the presence of anti-glycoprotein (GP) IIb/IIIa antibody at the concentration which completely inhibit platelet aggregation, ADP (0.5 – 3 μM) increased platelet adhesion to collagen in a concentration-dependent manner under static conditions when platelet-rich plasma (PRP) was used for the assay instead of washed platelets. This was also supported by the results of scanning electron microscopic analyses. The ADP-induced platelet adhesion to collagen was inhibited by PGI2 or beraprost, a stable analogue of PGI2, in a concentration-dependent manner (1 – 10 ng/ml). These findings suggested the presence of activation-dependent platelet adhesion to collagen. ADP-induced platelet adhesion to collagen was almost completely inhibited by anti-GPIa/IIa and anti-GPIIa antibodies. In the present study, we provide the first direct evidence that the activation-dependent platelet adhesion to collagen is induced by ADP and that GPIa/IIa also plays an important role in the mechanisms of this adhesion.

Prostacyclin and beraprost sodium (beraprost), a stable analogue of prostacyclin, increased cyclic AMP (cAMP) levels of cultured human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. The elevation of cAMP by beraprost was sustained longer than that by prostacyclin. The expression of thrombomodulin (TM) on membrane surface of HUVEC was enhanced by beraprost and prostacyclin, and the persistence of the increase in TM expression by beraprost was greater than prostacyclin. Dibutyryl cAMP (db-cAMP) mimicked the effects of beraprost and 3-isobutyl-1-methylxanthine enhanced the effects. Beraprost, prostacyclin and db-cAMP also effectively blocked the interleukin-1- and tumor necrosis factor-induced depression of TM expression substantially. These results suggest that TM expression is positively regulated by cAMP in HUVEC, and that beraprost may be potentially effective for reducing thrombotic events through the mechanism which initiates the stimulation of cAMP/TM system in vascular endothelial cells.

Beraprost sodium (beraprost) is a stable analogue of prostaglandin I2 (PGI2), which can be administrated orally. In the present study, the effect of beraprost on the activation process of polymorphonuclear leukocytes (PMNs) was examined in vitro. Beraprost effectively inhibited chemotaxis of PMNs induced by formyl-methionyl-leucyl-phenylalanine (FMLP). Like prostaglandin E2 (PGE2), beraprost elevated intracellular cAMP level and inhibited the influx of extracellular Ca2+ in PMNs. The concentration-response curves showed that the inhibitory effect of beraprost on chemotaxis was correlated with the increment of intracellular cAMP level of the PMNs and inhibition of influx of extracellular Ca2+. Beraprost also inhibited inositol phospholipid metabolic turnover and superoxide anion production of PMNs induced by FMLP at relatively high concentration. These results suggest that the inhibitory effect of beraprost on the PMN function especially chemotaxis is mediated through the elevation of the intracellular cAMP level, which interferes with the signal transduction process probably through the inhibition of Ca2+ mobilization in PMNs. The above-mentioned effects of beraprost were also the case with PGI2. The potency of beraprost was comparable to PGI2 in the present study. Considering its stability, these results thus raise a possibility that beraprost might exert anti-inflammatory effect in vivo.