In this study, the receptors and signals involved in collagen-induced platelet spreading were examined. It was found that platelet spreading on collagen (presenting a polygon shape with a number of filopodialike projections) was inhibited by the anti-integrin ␣ 2 antibody, suggesting the involvement of integrin ␣ 2 ␤ 1 in this process. Studies with a glutathione-Stransferase fusion protein that binds specifically to activated Rac and in vitro p21-activated kinase (PAK) kinase assays revealed that Rac and PAK were activated during this collagen-activated process. Platelet spreading on collagen-coated sur- IntroductionPlatelet adhesion to collagen fibers through specific receptors is one of the initial processes in thrombus formation. Platelets adhere to, then spread on, collagen fibers, and finally form aggregates by recruiting other platelets through glycoprotein (GP)IIb/IIIa-fibrinogen binding. Platelet spreading and aggregation appear to involve different processes. The collagen receptors responsible for each process also seem to be different. A number of candidates for platelet collagen receptors have been proposed to date, including integrin ␣ 2 ␤ 1 , GPVI, GPIV, and p65. 1 Among them, GPVI and integrin ␣ 2 ␤ 1 are accepted as collagen receptors necessary for collagen-induced platelet aggregation, because human blood platelets, which lack the surface expression of integrin ␣ 2 ␤ 1 2,3 or GPVI, 4,5 fail to form aggregates on collagen stimulation. The GPVI-mediated activation signals that lead to platelet aggregation have been extensively investigated and several signaling molecules have been characterized. [6][7][8] However, whether integrin ␣ 2 ␤ 1 mediates activation signals during platelet aggregation remains controversial.There have been several studies on the receptors responsible for platelet spreading on collagen-coated surfaces. Kehrel et al 9 reported that only 9.9% of GPIa-deficient platelets spread on type I collagen, in contrast to 82% with control platelets. Nakamura et al 10 showed that an anti-integrin ␣ 2 ␤ 1 antibody markedly inhibited platelet spreading on collagen, whereas an anti-GPIV antibody had virtually no effect. They also reported that an anti-GPVI antibody had only slight inhibitory effects on platelet spreading, although Falet et al 30 have recently shown that human platelets spread on collagen-related peptide (CRP)-coated surfaces, implying that signals from GPVI can also mediate platelet spreading. These findings suggest that integrin ␣ 2 ␤ 1 is at least one of the major receptors responsible for platelet spreading and that it elicits certain signals necessary for this spreading. However, as described above for platelet aggregation, the presence of integrin ␣ 2 ␤ 1 -mediated activation signals has been challenged by others. 1,11 Thus, we sought to characterize the integrin ␣ 2 ␤ 1 -mediated signals that lead to platelet spreading in this study. Platelets adhere to collagen, then extend projections, and finally form curtainlike extensions between the projections during the proces...

Activation of blood cells during hemodialysis is considered to be a significant determinant of biocompatibility of the hemodialysis membrane because it may affect patient health adversely through microvascular inflammation and oxidative stress. This study found very different cell activation among various polysulfone (PSf) hemodialysis membranes. For example, CX‐U, a conventional PSf membrane, induced marked adhesion of platelets to its surface and increased surface expression of activated CD11b and production of reactive oxygen species (ROS) by neutrophils; while NV‐U, a hydrophilic polymer‐immobilized PSf membrane, caused little platelet adhesion and slight CD11b expression and ROS production by neutrophils. Analysis of the molecular mechanisms of the above phenomena on CX‐U and NV‐U indicated that anti‐integrin GPIIb/IIIa antibody blocked platelet adhesion, and that the combination of anti‐CD11b (integrin α subunit of Mac‐1) and anti‐integrin αvβ3 antibodies blocked ROS production by neutrophils. Plasma‐derived fibrinogen, a major ligand of GPIIb/IIIa, Mac‐1, and αvβ3 on membranes, was thus analyzed and found to be more adsorbed to CX‐U than to NV‐U. Moreover, comparison between five PSf membranes showed that the number of adherent platelets and neutrophil ROS production increased with increasing fibrinogen adsorption. These results suggested that fibrinogen, adsorbed on membranes, induced GPIIb/IIIa‐mediated platelet activation and Mac‐1/αvβ3‐mediated neutrophil activation, depending on the amount of adsorption. In conclusion, the use of biocompatible membranes like NV‐U, which show lower adsorption of fibrinogen, is expected to reduce hemodialysis‐induced inflammation and oxidative stress by minimizing cell activation.

Although glycoprotein Ia/IIa (GPIa/IIa, integrin ␣ 2 ␤ 1 ) has established its role as a collagen receptor, it remains unclear whether GPIa/IIa mediates activation signals. In this study, we show that rhodocytin, purified from the Calloselasma rhodostoma venom, induces platelet aggregation, which can be blocked by anti-GPIa monoclonal antibodies. Studies with rhodocytin-coupled beads and liposomes loaded with recombinant GPIa/IIa demonstrated that rhodocytin directly binds to GPIa/IIa independently of divalent cations. In vitro kinase assays and Western blotting of GPIa immunoprecipitates revealed that Src and Lyn constitutively associate with GPIa/IIa and that Src activity increases transiently after rhodocytin stimulation. Src specifically associates with p130 Crk-associated substrate (Cas) in a manner dependent upon Cas phosphorylation, suggesting that Src is responsible for Cas tyrosine phosphorylation. While all these phenomena occur early after rhodocytin stimulation in a cAMP-resistant manner, tyrosine phosphorylation of Syk and phospholipase C␥2, intracellular Ca 2؉ mobilization, and platelet aggregation occur later in a cAMP-sensitive manner. Cytochalasin D, which interferes with actin polymerization and blocks receptor clustering, inhibits all the rhodocytin-mediated signals we examined in this study. We suggest that rhodocytin, by clustering GPIa/IIa, activates GPIa/IIa-associated Src, which then mediates downstream activation signals.

Liposomes carrying both recombinant glycoprotein Ia/IIa (rGPIa/IIa) and Ib␣ (rGPIb␣) (rGPIa/IIa-Ib␣-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␣ alone on the VWF surface. In the absence of soluble VWF, the adhesion of rGPIa/IIa-Ib␣-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␣ densities of 2.17 ؋ 10 3 and 1.00 ؋ 10 4 molecules per particle, respectively, was efficient at high shear rates, reduction in rGPIb␣ density to 5.27 ؋ 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␣ (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␣-liposomes to the collagen surface at low shear rates, while the rGPIa/IIa-collagen and rGPIb␣-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. IntroductionThe basic and important platelet functions for primary hemostasis are adhesion and aggregation, and this can be easily understood from the observations that patients with congenital platelet membrane defects such as Bernard-Soulier syndrome or Glanzmann thrombasthenia are deficient in platelet adhesion or aggregation and have severe bleeding tendencies. The contribution of specific platelet receptors or adhesive proteins to platelet adhesion and aggregation onto immobilized collagen under flow conditions is usually studied with monoclonal antibodies or inhibitors specific to particular platelet receptors or adhesive proteins or, also, with blood from patients with congenital bleeding disorders deficient in specific receptors or adhesive proteins. These analyses indicate that initial platelet adhesion depends on the interaction of glycoprotein (GP) Ib/IX/V complexes on platelets with von Willebrand factor (VWF) adsorbed on the collagen surface. This is a rapid but low-affinity interaction, suggesting that it serves to tether platelets, flowing at high speed in the bloodstream, to the collagen surface. [1][2][3][4] The collagen receptors of the tethered platelets then bind strongly with the collagen surface, activating platelets to form aggregates. This was supported by observations that platelets ...