Edward F. Plow

Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif

The tripeptide Arg-Gly-Asp (RGD) was originally identified as the sequence within fibronectin that mediates cell attachment. The RGD motif has now been found in numerous other proteins and supports cell adhesion in many, but not all, of these. The integrins, a family of cell-surface proteins, act as receptors for cell adhesion molecules. A subset of the integrins recognize the RGD motif within their ligands, the binding of which mediates both cell-substratum and cell-cell interactions. RGD peptides and mimetics, in addition to providing insights into the fundamental mechanisms of cell adhesion, are potential therapeutic agents for the treatment of diseases such as thrombosis and cancer.

Ligands "activate" integrin αIIbβ3 (platelet GPIIb-IIIa)

Abstract Integrin α IIb β 3 (platelet GPllb-Illa) binds fibrinogen via recognition sequences such as Arg-Gly-Asp (RGD). Fibrinogen binding requires agonist activation of platelets, whereas the binding of short synthetic RGD peptides does not. We now find that RGD peptide binding leads to changes in α IIb β 3 that are associated with acquisition of high affinity fibrinogen-binding function (activation) and subsequent platelet aggregation. The structural specificities for peptide activation and for inhibition of ligand binding are similar, indicating that both are consequences of occupancy of the same site(s) on α IIb β 3 Thus, the RGD sequence is a trigger of high affinity ligand binding to α IIb β 3 and certain RGD-mimetics are partial agonists as well as competitive antagonists of integrin function.

The fibrinolytic system in man.

The fibrinolytic system comprises a proenzyme, plasminogen, which can be activated to the active enzyme plasmin, that will degrade fibrin by different types of plasminogen activators. Inhibition of fibrinolysis may occur at the level of plasmin or at the level of the activators. Fibrinolysis in human blood seems to be regulated by specific molecular interactions between these components. In plasma, normally no systemic plasminogen activation occurs. When fibrin is formed, small amounts of plasminogen activator and plasminogen adsorb to the fibrin, and plasmin is generated in situ. The formed plasmin, which remains transiently complexed to fibrin, is only slowly inactivated by alpha 2-antiplasmin, while plasmin, which is released from digested fibrin, is rapidly and irreversibly neutralized. The fibrinolytic process, thus, seems to be triggered by and confined to fibrin. Thrombus formation may occur as the result of insufficient activation of the fibrinolytic system and (or) the presence of excess inhibitors, while excessive activation and/or deficiency of inhibitors might cause excessive plasmin formation and a bleeding tendency. Evidence obtained in animal models suggests that tissue-type plasminogen activator, obtained by recombinant DNA technology, may constitute a specific clot-selective thrombolytic agent with higher specific activity and fewer side effects than those currently in use.

The major fibrinolytic proteases of human leukocytes

The major fibrinolytic enzymes present in leukocyte granules and active at physiological pH have been identified. The fibrinolytic activity in extracts of leukocyte granules was found to fibrinogen-Sepharose and eluted with 8.0 M urea. Two distinct zones of fibrinolytic activity were detected upon electrophoresis of leukocyte extracts on fibrinogen polyacrylamide gels, and both were qualitatively recovered in the 8.0 M urea eluate. Quantitatively, greater than 95% of the fibrinolytic activity was recovered in the urea eluate. Two major leukocyte proteases, elastase (EC 3.4.21.11) and cathepsin G (EC 3.4.21.-), were quantitatively recovered in the urea eluate. Both enzymes, when purified separately by affinity chromatography, were shown to: (a) possess fibrinolytic activity; (b) coincide in mobility and generate the two zones of fibrinolytic activity on fibrinogen polyacrylamide gels; and (c) quantitatively reconstitute the fibrinolytic activity of the leukocyte granules when combined at activity levels present in granular extracts. A highly significant correlation (r = 0.98) was found between the fibrinolytic activity and the sum of elastase and cathepsin G activity in leukocytes from five donors. Thus, elastase and cathepsin G are the major enzymes of the leukocyte fibrinolytic pathway, and fibrinogen-Sepharose chromatography may be used to obtain these enzymes.

Radiographic contrast media infusions. Measurement of histamine, complement, and fibrin split products and correlation with clinical parameters.

Abstract To further an understanding of the effects of radiographic contrast media (RCM) infusions, 43 patients underwent clinical evaluation, including allergy history and skin testing, at least 48 hr before receiving a bolus of Renografin-60 (meglumine diatrizoate [52] and sodium diatrizoate [8]) for intravenous pyelography. Venous plasma samples were obtained serially before and at 2, 4, 10, 30, and 60 min after the infusion. Each sample was assayed for histamine by isotopic enzyme assay, for complement by total hemolytic activity, and for fibrin split products by radioimmunoassay. Immediate generalized reactions occurred in 6 of the patients. Each of the 43 patients had a change in at least 1 of the mediators measured. Plasma histamine rose in 40% of the patients (mean, 3.1 ± 1.62 ng/ml). Plasma complement hemolytic activity decreased in 63% (mean, 29 ± 19%). Fibrin split products were detected in 41%. In comparing the 6 patients who experienced reactions to the 37 who did not, there were no statistically significant differences in rise in plasma histamine, decrease in complement activity, presence of fibrin split products, personal history of allergy, prior exposure or reaction to RCM, skin tests to RCM, and histamine or saline. Although histamine levels, complement activity, and fibrin split products changed in a substantial number of patients undergoing RCM infusions, these changes did not correlate with immediate generalized reactions or with the previously described clinical parameters. It is probable that other modulating factors play significant roles in determining whether or not a reaction occurs.

THE FIBRINOGEN‐DEPENDENT PATHWAY OF PLATELET AGGREGATION

Fibrinogen participates in platelet aggregation induced by ADP and this participation is receptor mediated. The role of fibrinogen in platelet function is not restricted to the ADP stimulus as the molecule may regulate aggregation induced by the direct interaction of the protein with a single, specific receptor system. Thus, a common fibrinogen-dependent mechanism leading to platelet aggregation is hypothesized.

Plasminogen receptors in the mediation of pericellular proteolysis

A wide variety of cells bind plasminogen with very high capacity, with similar affinity and recognize the same structural features within the plasminogen molecule. As a consequence of binding to cell surfaces, plasminogen is more readily activated to plasmin. Plasmin remains cell-bound where it can degrade matrix constituents and is protected from inactivation by alpha 2-antiplasmin. Thus, the functional consequence of plasminogen binding to cells is pericellular proteolysis, permitting cell migration. Both proteins and nonprotein cell-surface constituents function as plasminogen binding sites. Gangliosides exhibit the appropriate properties of the non-protein plasminogen receptors.

The contribution of leukocyte proteases to fibrinolysis

SummaryPolymorphonuclear leukocytes accumulate within blood clots and may contribute to fibrinolysis. The primary fibrinolytic enzymes of neutrophils are cathepsin G and elastase. Fibrin can be exposed to these granular enzymes as a result of cell lysis, phagocytosis of fibrin, or secretion of the enzymes from the cells. Neutrophil secretion occurs in association with blood coagulation and is dependent upon a plasma factor(s) and calcium. After secretion, the enzymes can degrade fibirn within a plasma environment. This is demonstrated by the inhibition of fibrinolysis by specific inhibitors of elastase and the augmentation of fibrinolysis by neutralization of the primary plasma inhibitor of elastase, α1-proteinase inhibitor. A radioimmunoassay which discriminates elastase from plasmic degradation products of fibrinogen has been developed. In this assay, elastase elicited degradation products of fibrin(ogen) were detected in certain pathophysiologic plasma samples. Taken together, these findings indicate a role for leukocyte proteases in physiological fibrinolysis.

Human monoclonal autoantibody 2E7 is specific for a peptide sequence of platelet glycoprotein IIb. Localization of the epitope to IIb231–238 with an immunodominant Trp235

2E7 is a human monoclonal IgM autoantibody that binds to a site on the heavy chain of the human platelet integrin alpha subunit glycoprotein IIb. The epitope recognized by 2E7 is stable to denaturation with sodium dodecyl sulfate and reduction of disulfide bonds but is destroyed by proteolysis with papain, chymotrypsin or elastase. By evaluating the reaction of 2E7 with a number of protein sequences from the IIb heavy chain, we have determined that the epitope is located in the octapeptide Phe-Asp-Gly-Tyr-Trp-Gly-Tyr-Ser (FDGYWGYS), corresponding to residues 231-238, and that substitution of the Trp at position 235 completely destroys the epitope. This represents the first precise localization of an epitope on the human platelet integrin IIb-IIIa or on any platelet membrane glycoprotein that is recognized by a human autoantibody.

Polymorphism of platelet glycoprotein Ib in the united states

Platelet glycoprotein (GP) Ib from healthy individuals from the United States was analyzed using SDS-polyacrylamide gel electrophoresis and staining with peanut agglutinin-coupled peroxidase or wheat germ agglutinin-coupled peroxidase after transfer of electrophoresed proteins to nitrocellulose. The GPIb from American Caucasians (109 individuals) was found to be polymorphic, showing types A, B, C, and D of GPIb, similar to what was observed in Japanese. The phenotype distribution pattern, however, was very different from that observed in the Japanese population. GPIb heterogeneity was also observed in American Blacks, Asian Americans, and Americans of Hispanic origin. Thus, it appears that the polymorphism of GPIb is a general phenomenon which should be regarded as an important factor when doing studies on this glycoprotein.