Lawrence L. K. Leung

Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen.

We have recently reported the isolation of purified platelet membrane glycoproteins IIb and IIIa and the generation of monospecific antisera to these membrane proteins. Using these monospecific antisera in an enzyme-linked immunosorbent assay system, it is no demonstrated that glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a complex with purified human fibrinogen. The formation of this GPIIb-GPIIIa fibrinogen complex is calcium dependent, fibrinogen specific, saturable, and inhibited by specific amino sugars and amino acids. These observations suggest that the GPIIb-GPIIIa macromolecular complex on the platelet surface acts under the proper physiologic circumstances as the fibrinogen binding site required for normal platelet aggregation.

Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator.

Thrombospondin (TSP), a multifunctional alpha-granule glycoprotein of platelets, binds fibrinogen, fibronectin, heparin, and histidine-rich glycoprotein and thus may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a complex with purified human plasminogen (Plg). Complex formation was detected by rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins. Significant complex formation of fluid-phase Plg with adsorbed TSP was also demonstrated by enzyme-linked immunosorbent assay (ELISA). The complex formation was specific, saturable, and inhibited by excess fluid-phase TSP, with an apparent KD of approximately 35 nM. In both ELISA and rocket immunoelectrophoresis systems, complex formation was inhibited by 10 mM epsilon-amino-n-caproic acid, implying that there is a role for the lysine binding sites of Plg in mediating the interaction. TSP also formed a complex with plasmin as detected by ELISA but did not directly inhibit plasmin activity measured with a synthetic fluorometric substrate or with a 125I-fibrin plate assay. TSP, when incubated with Plg before addition to 125I-fibrin plates significantly inhibited the generation of plasmin activity by tissue plasminogen activator (TPA) in a manner that was calcium dependent. A kinetic study of Plg activation by TPA in the presence of TSP demonstrated that Michaelis-Menten kinetics were followed and that TSP acted as a noncompetitive inhibitor. These studies support the hypothesis that TSP, acting as a multifunctional regulator in focal areas of active hemostasis, could serve as a prothrombotic influence, leading to increased deposition of fibrin.

Complex formation of platelet thrombospondin with histidine-rich glycoprotein.

Thrombospondin and histidine-rich glycoprotein are two proteins with diverse biological activities which have been associated with human platelets and other cell systems. Using an enzyme-linked immunosorbent assay, we have demonstrated that purified human platelet thrombospondin formed a complex with purified human plasma histidine-rich glycoprotein. The formation of the thrombospondin-histidine-rich glycoprotein complex was specific, concentration dependent, and saturable. Significant binding was detected when histidine-rich glycoprotein was incubated with thrombospondin immobilized on anti-thrombospondin IgG-coated plates, indicating that the observed complex formation was not due to a thrombospondin interaction with the plastic surface. Sucrose-density-gradient ultracentrifugation of a mixture of thrombospondin and histidine-rich glycoprotein also revealed the formation of fluid-phase complexes, with an estimated stoichiometry of 1 thrombospondin: 3.5 histidine-rich glycoprotein. Fibrinogen, which has been previously shown to bind to absorbed thrombospondin, did not inhibit the formation of the thrombospondin-histidine-rich glycoprotein complex. Histidine-rich glycoprotein complexed with thrombospondin was capable of binding heparin and neutralizing the anticoagulant activity of heparin in plasma. Specific complex formation between thrombospondin and histidine-rich glycoprotein may play a significant role in influencing platelet blood vessel wall interactions as well as modulating the association of various cells with the extracellular matrix.

Platelet thrombospondin forms a trimolecular complex with plasminogen and histidine-rich glycoprotein.

Thrombospondin (TSP), a multifunctional alpha-granule glycoprotein of human platelets binds fibrinogen, fibronectin, heparin, histidine-rich glycoprotein (HRGP), and plasminogen (Plg), and thus, may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a trimolecular complex with human Plg and HRGP. Complex formation was detected by a specific binding enzyme-linked immunosorbent assay (ELISA) which demonstrated simultaneous binding of fluid-phase Plg and HRGP to TSP adsorbed to microtitration wells. While neither ligand inhibited complex formation of the other with TSP, 10 mM epsilon-amino-n-caproic acid selectively blocked incorporation of Plg into the complex, suggesting that TSP contains independent binding sites for Plg and HRGP. Comparable extent of trimolecular complex formation was also detected when TSP monomer was substituted for whole TSP in the ELISA. HRGP covalently cross-linked to Sepharose 4B simultaneously bound both 125I-TSP and 131I-Plg, confirming trimolecular complex formation. Rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins into anti-Plg containing agarose also confirmed trimolecular complex formation. The TSP-HRGP-Plg complex bound a similar amount of heparin as the TSP-HRGP complex, demonstrating that the HRGP within the trimolecular complex maintained functional capability. Similarly, using a fluorometric plasmin substrate, the trimolecular complex was shown to be an effective substrate for tissue plasminogen activator. Significant amounts of plasmin were generated from the TSP-HRGP-Plg complex (equivalent to that from the TSP-Plg complex), but the rate of plasmin generation from the trimolecular complex was greater than from the bimolecular complex, suggesting an important interaction of HRGP with Plg when both are complexed to TSP. The macromolecular assembly of these three proteins on cellular surfaces, such as the platelet, may serve important regulatory functions, both prothrombotic at sites of active fibrin deposition and proteolytic in nonfibrin-containing microenvironments.

Molecular Mechanisms of Platelet Adhesion and Platelet Aggregation

Certain primitive cell systems, such as cellular slime molds, exist either in unicellular vegetative forms or in a differentiated state in which they become adhesive and aggregate into a multicellular structure. An example of this mechanism of cellular behavior is shown in Figure 1, which illustrates the life cycle of Acrasis rosea, an acrasid cellular slime mold. The uninucleate ameboid cells move, divide by binary fission, deplete the local environment of the food supply, aggregate, and form fruiting bodies. The mechanisms associated with the development of the adhesive state have been studied most extensively in the dictyostelid cellular slime molds. When the cells of this species become adhesive, they synthesize surface polyvalent carbohydrate-binding proteins or lectins that mediate cell to cell adhesion (Frasier and Glaser, 1979; Barondes, 1981). This stage of the cell cycle is associated with activation of a large number of new genes (Blumberg et al., 1982), presumably coding for cell-surface proteins that mediate the conversion to the adhesive state. Cell adhesion culminating in aggregation takes place via the interaction of carbohydrate-binding sites of a cell-surface lectin with oligosaccharides on membrane glycoprotein receptors. The initial interaction of one lectin molecule with one receptor oligosaccharide followed by binding at multiple sites leads to rapid and stable cohesion (aggregation). Discoidin I and II, two closely related galactose-binding lectins synthesized by Dictyostelium dis coideum during the conversion from the noncohesive to the aggregating cohesive stage, may function at different stages of the aggregation process by binding to different oligosaccharide-containing receptors at the cell surface (Berger and Armant, 1982). Thus, at least in primitive systems, the aggregation process appears to involve the sequential exposure of a family of cell-surface recognition molecules, some of which serve as lectins. It is probable that human platelets may recapitulate some of these primitive cellular responses during the process of agonist-induced aggregation.

[23] Platelet histidine-rich glycoprotein

Publisher Summary This chapter describes the purification and assay of plasma histidine-rich glycoprotein (HRGP). The chapter presents a study for the purification of plasma HRGP, during which soybean trypsin inhibitor was added to acid–citrate–dextrose (ACD) plasma. Fifty percent polyethylene glycol was added to a final concentration of 6 percent to remove the fibrinogen and precipitate by centrifugation. The supernatant was processed by adsorption to CM-cellulose, elution with NH 4 HCO 3 , and affinity chromatography of the eluate on a column containing the high-affinity lysine-binding site of plasminogen. Traces of IgG, fibrinogen, and plasminogen were detected in the HRGP preparation by enzyme-linked immunosorbent assays. These were removed by affinity chromatography utilizing the insolubilized rabbit IgG antisera directed against these contaminants. Purified plasma HRGP served as a standard. To investigate the immunochemical characteristics of the platelet HRGP, purified plasma HRGP and thrombin-stimulated platelet releasate were analyzed using the monospecific anti-HRGP on electrophoretic blots.