The interactions between ligands containing the recognition sequence arginine-glycine-aspartic acid (RGD) and integrin receptors are important in many cell-cell and cell-protein interactions. The platelet contains five integrin receptors and they contribute significantly to platelet adhesion and aggregation. To investigate the RGD binding domains on platelet integrins, we immobilized a series of RGD peptides containing variable numbers of glycine residues [(G)n-RGDF] on polyacrylonitrile beads and evaluated the ability of the beads to interact with platelets. With native platelets, virtually no interaction occurred with G1-RGDF beads, but the interactions increased as the number of glycine residues increased, plateauing with the G9- RGDF and G11-RGDF beads. ADP pretreatment enhanced the interactions with all of the beads, whereas prostaglandin E1 pretreatment eliminated the interactions with the shortest peptide beads, but only partially inhibited interactions with the longer peptide beads. Monoclonal antibodies to glycoprotein (GP) IIb/IIIa were most effective in inhibiting the interactions, but antibodies to GPIIb/IIIa with similar inhibitory effects on fibrinogen binding varied dramatically in their ability to inhibit the interaction between platelets and immobilized RGD peptides. Our data indicate that the majority of RGD binding sites on GPIIb/IIIa can be reached by peptides that extend out approximately 11 to 32 A from the surface of the bead, and these results are in accord with the dimensions of integrin receptors deduced from electron microscopy. Activation of GPIIb/IIIa facilitates the interactions, but platelet inhibition fails to eliminate the interactions with the longer peptide beads, suggesting that access to the RGD binding site on at least a fraction of the GPIIb/IIIa receptors is always possible for preferred ligands. Finally, we found that the G3-RGDF peptide beads were uniquely sensitive to the activation state of the GPIIb/IIIa receptor.

Platelet glycocalicin (GC) is the extramembranous portion of GPIb alpha that can be rapidly cleaved by enzymes such as calpain, plasmin, trypsin, elastase, etc. Quantitative cleavage will ultimately result in an acquired Bernard-Soulier-like bleeding disorder, and circulating GC may act as a potential inhibitor of platelet adhesion. We have developed and standardized a new enzyme-linked immunosorbent assay (ELISA), which uses two monoclonal antibodies (mAbs), both of which bind to the amino-terminal 45-kD fragment of GC and inhibit platelet- von Willebrand interactions and the streptavidin-biotin system. First, the methodology was evaluated and standardized with special emphasis on the anticoagulant and the inhibitors (EDTA, prostaglandin E1 [PGE1], aprotinin, N-ethyl-maleimide), the mode of high-speed centrifugation (to avoid platelet microparticles), and the standards used (purified GPIb and GC). This assay was then used to analyze the GC levels of healthy subjects (2.04 +/- 0.46 micrograms/mL) and of patients with selected diseases. The results of patients with aplastic anemia and thrombocytosis confirmed that GC levels are clearly dependent on the platelet count, which was the basis for the introduction of the GC index, the standardization of GC for a platelet count of 250 x 10(9)/L. The GC index discriminates reliably patients with active immune thrombocytopenic purpura from those in remission. GC levels are elevated in patients on hemodialysis (3.62 +/- 0.75 micrograms/mL, P < .001). The high GC index (6.93 +/- 4.21, P < .001) in cirrhosis patients suggests an increased platelet turnover and/or abnormal proteolysis. In contrast to other groups, we have not found that recombinant tissue plasminogen activator (rtPA) treatment of patients with myocardial infarction increases GC levels. However, concentrations are elevated in leukemia and the highest levels found are approximately 40 micrograms/mL. These studies suggest that GC is a useful platelet marker in certain diseases, which directly reflects platelet damage and possibly platelet dysfunction.

To evaluate the physiologic importance of the different collagen receptors on platelets, we screened 806 patients admitted to the hospital because of hemorrhagic diathesis for eventual laboratory evidence of a pathologic platelet collagen interaction, and found 5 patients with an isolated deficiency in collagen-induced platelet aggregation. Four of these five patients had a partial defect, one had a complete defect. The structural and functional analysis of the platelets from the patient with a complete defect showed a deficiency in glycoprotein (GP) IV and autoantibodies against GPIIb/IIIa, GPIa/IIa, and GPIV. Patient plasma had only a minimal effect on normal control platelets and Naka-negative platelets. The analyses of the defect in the patient and of the data in the literature suggest that a single defect may not result in clinical bleeding (GPIV-deficient patients do not bleed), but may become symptomatic in combination with another defect such as the autoantibodies against GPIa/IIa, GPIV, and/or GPIIb/IIIa, all of which are involved in platelet collagen interactions (three of four of our immune thrombocytopenic purpura patients with anti-GPIV and anti-GPIIb/IIIa autoantibodies had a bleeding disorder). We hypothesize that it is the synergism of two abnormalities that results in the defective function, a mechanism that is in agreement with earlier studies on platelet collagen interaction that suggests that a double defect in platelet collagen interactions is required to become clinically apparent.

Using intact human platelets as the immunogen and a functional, collagen-coated bead agglutination assay, we have produced a murine monoclonal antibody (6F1) that blocks the interaction between platelets and collagen in the presence of Mg++. 6F1 affinity-purified the platelet glycoprotein Ia/IIa complex, and approximately 800 molecules of 6F1 bound per platelet at saturation. 6F1 nearly completely inhibited collagen-induced platelet aggregation and inhibited platelet adhesion to collagen by greater than 95% when plasma proteins were absent. Antibody 10E5, which blocks the binding of adhesive glycoproteins to GPIIb/IIIa, produced only minor inhibition (approximately 25%) of adhesion under the same circumstances. In contrast, when tested in platelet-rich plasma (PRP), 6F1 had only a minor effect on collagen-induced platelet aggregation, prolonging the lag phase but not the slope or maximum aggregation. Similarly, when collagen was precoated with plasma, 6F1 caused less inhibition of platelet adhesion (53%) than without the precoating (greater than 95%). Antibody 10E5 inhibited this adhesion by 32%, and the combination of 6F1 and 10E5 was more effective than either alone, inhibiting it by 90%. Time course studies of platelet agglutination of collagen-coated beads using PRP containing physiologic concentrations of divalent cations showed early inhibition by 6F1, indicating that the GPIa/IIa receptor operates in this environment. With more prolonged incubation, however, 6F1 was less effective; this later agglutination could be partially prevented by adding 10E5 or PGE1 to the 6F1. These data support a model wherein collagen can directly interact with GPIa/IIa and can indirectly interact with GPIIb/IIIa via intermediary adhesive proteins. The physiological significance of these interactions, and potential interactions with other receptors, remains to be established.