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E F Plow

Publications and source records attributed to E F Plow.

At least 145 records · Page 8Linked to original sources
PubMed1989

Efficient surface expression of platelet GPIIb-IIIa requires both subunits.

Platelet membrane GPIIb-IIIa is a member of the integrin family of heterodimeric adhesion receptors. Processing and export of certain leukocyte and melanoma integrins is disrupted in cells lacking one subunit. We found that surface expression of GPIIb-IIIa, measured by fluorescent activated cell sorting or by surface labeling, required cotransfection of both subunits. In contrast, surface expression was not detected when the subunits were transfected individually. Immunoprecipitation of metabolically labeled transfected cells confirmed the presence of comparable levels of intracellular protein in all cases. When both subunits were transfected, post-translational cleavage of Pro-GPIIb to yield GPIIb heavy chain was also seen, while transfection with GPIIb alone resulted in coprecipitation of Pro-GPIIb with a second band that may be an endogenous beta subunit. Pro-GPIIb in these transfectants was not processed to yield GPIIb heavy chain. When transfected into COS cells alone, transiently expressed GPIIIa remained intracellular and did not appear to complex with any endogenous proteins. Thus, surface expression of processed GPIIb-IIIa depends on the presence of both subunits; the coordinate reduction of both subunits observed in some cases of Glanzmann's thrombasthenia may result from mutation affecting only one.

Animals↗
PubMed1988

Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor.

Many adhesive interactions are mediated by Arg-Gly-Asp (RGD) sequences within adhesive proteins. Such RGD sequences are frequently recognized by structurally related heterodimers that are members of the integrin family of adhesion receptors. A region was found in the platelet RGD receptor, gpIIb/IIIa, to which an RGD peptide becomes chemically cross-linked. This region corresponds to residues 109 to 171 of gpIIIa. This segment is conserved among the beta subunits of the integrins (76 percent identity of sequence), indicating that it may play a role in the adhesive functions of this family of receptors.

Amino Acid Sequence↗
PubMed1988

Occupancy of an adhesive glycoprotein receptor modulates expression of an antigenic site involved in cell adhesion.

Binding of ligands that contain Arg-Gly-Asp to adhesion receptors induces cell spreading and aggregation and alters gene expression, possibly due to conformational changes within occupied adhesion receptors. PMI-1 is a monoclonal antibody which reacts with the platelet fibrinogen receptor, glycoprotein IIb-IIIa, and reports such a conformational change. ADP stimulation of platelets results in a fibrinogen-dependent increase in binding of the PMI-1 antibody. Peptides containing Arg-Gly-Asp also reversibly increase the binding of this antibody to cells and to purified glycoprotein IIb-IIIa. The PMI-1 antibody inhibits platelet adhesion and spreading on certain substrata (Shadle, P. J., Ginsberg, M. H., Plow, E. F., and Barondes, S. H. (1984) J. Cell Biol. 99, 2056-2060); thus this occupancy-modulated site may participate in adhesive function.

Adenosine Diphosphate↗
PubMed1988

The cell-binding domains of plasminogen and their function in plasma.

Plasminogen binding sites are expressed by a wide variety of cell types and serve to promote fibrinolysis and local proteolysis. In this study, the recognition specificity of cells for plasminogen has been examined, primarily using platelets as models. Analyses with plasminogen fragments implicated residues 79-337 (or 353), comprising the first three kringles of plasminogen, as a primary recognition site for plasminogen binding to both thrombin-stimulated and nonstimulated platelets. Other regions of plasminogen, namely residues 354-439 and 442-790, can also participate in the interaction, and these other regions contribute differentially to the binding of the ligand to stimulated and nonstimulated platelets. Binding to nucleated cells, with U937 cells serving as the prototype, is dependent upon a recognition specificity similar to that of unstimulated platelets. Binding of Glu-plasminogen, the native form of the molecule, to thrombin-stimulated platelets has been shown previously to require platelet fibrin. By comparing the interaction of Glu-plasminogen and its degradation product, Lys-plasminogen, with thrombin-stimulated platelets, it is concluded that the cell surface uniquely enhances the affinity of Glu-, but not Lys-plasminogen, for fibrin. Finally, we have demonstrated that cellular receptors and interactive sites within plasminogen are available in the plasma environment. Thus, the functions ascribed to cellular plasminogen receptors can occur within a physiologic setting.

Binding Sites↗
PubMed1988

Alternative proteolytic processing of platelet membrane glycoprotein IIb.

Platelet membrane glycoprotein (GP) IIb-IIIa is a component of a receptor for the adhesive proteins fibrinogen, fibronectin, and von Willebrand factor. GPIIb is initially synthesized as a single-chain polypeptide that is proteolytically processed to yield the two chains of mature GPIIb present on the cell surface. Analysis of the amino acid sequence surrounding the proposed light-heavy chain junction of GPIIb suggests a second potential site following a pair of basic residues 12-15 residues upstream from the reported amino terminus of the light chain. We have utilized anti-peptide antibodies to examine the possibility of alternative cleavage at these two potential sites. Peptide V43 precedes the dibasic sequence and is known to reside in the heavy chain. Peptide V41 contains the sequence between the two potential sites. In immunoblots, anti-V43 reacted only with the heavy chain while anti-V41 reacted only with the light chain. Immunoprecipitation of surface-labeled platelets indicated 97% of the GPIIb light chain contains the V41 sequence while approximately 3% of GPIIb molecules lack the V41 sequence on both the light and heavy chains. These data indicate that GPIIb is primarily cleaved 12-15 amino acids upstream from the reported amino terminus of the light chain while in a minor proportion of GPIIb molecules cleavage occurs at both sites.

Amino Acid Sequence↗
PubMed1988

Neutrophil secretion during blood coagulation: evidence for a prekallikrein independent pathway.

In association with blood coagulation, neutrophils undergo a secretory response (Plow, J Clin Invest 69: 564, 1982) and it has been suggested that plasma kallikrein is responsible for inducing this reaction (Wachtfogel et al., J Clin Invest 72: 1672, 1983). To assess the contribution of kallikrein to this response, the capacity of normal and prekallikrein-deficient blood and plasma to support secretion has been compared utilizing elastase as a marker of secretion. Serial dilutions of prekallikrein-deficient plasma were as effective as normal plasma in supporting neutrophil release of elastase. The extent of elastase release in spontaneously clotting normal and prekallikrein-deficient blood was similar. At 37 degrees C in whole blood or at 22 degrees C in plasma, prekallikrein activators had the same effect in neutrophil secretion in normal and prekallikrein-deficient blood and plasma samples. Taken together, these results provide evidence for the existence of a prekallikrein independent pathway that can function as a predominant mechanism for induction of neutrophil secretion during blood coagulation.

Blood Coagulation↗
PubMed1988

Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets.

A chemical cross-linking approach has been used to characterize the interaction of platelets with small peptides of 7 and 14 residues containing the arginyl-glycyl-aspartic acid (RGD) sequence recognized by a variety of cellular adhesion receptors. The radioiodinated peptides were bound to platelets, and chemical cross-linking was attained by subsequent addition of bifunctional reagents. Three different cross-linking reagents coupled the RGD-containing peptides to platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa), and both subunits of this platelet membrane glycoprotein became radiolabeled with the RGD peptides. Platelet stimulation with agonists including thrombin, phorbol myristrate acetate, and ADP increased the extent of cross-linking by predominantly enhancing the coupling of the RGD peptides to the GPIIIa subunit. Cross-linking of the labeled RGD peptides to GPIIb and GPIIIa on stimulated and nonstimulated platelets exhibited structural specificity and was inhibited by excess nonlabeled RGD peptides. The interactions were inhibited by nonlabeled RGD peptides and a peptide with an amino acid sequence corresponding to the carboxyl terminus of the gamma chain of fibrinogen but less effectively by an arginyl-glycyl-glutamic acid peptide. Cross-linking of the RGD peptides to GPIIb-IIIa was divalent ion-dependent and, on stimulated platelets, was inhibited by the adhesive proteins fibrinogen and fibronectin, but not by albumin. These results indicate that the RGD-binding sites on platelets reside in close proximity to both subunits of GPIIb-IIIa and that platelet stimulation alters the topography of these sites such that the peptides become more efficiently cross-linked to GPIIIa.

Amino Acid Sequence↗
PubMed1988

Plasminogen receptors, urokinase receptors, and their modulation on human endothelial cells.

Endothelial cells are centrally involved in regulation of fibrinolysis, and receptors for plasminogen and urokinase provide a mechanism by which cells can regulate their fibrinolytic function. Therefore, the existence and characteristics of receptors for these fibrinolytic components on cultured human umbilical vein endothelial cells were examined. We verified the presence of plasminogen receptors on these cells (Kd = 2.1 +/- 1.3 mumol/L, and 1.8 +/- 1.3 x 10(7) binding sites/cell). These binding parameters and other characteristics indicate that these receptors are closely related to the plasminogen receptors on many circulating and adherent cells. Specific binding sites that interact with two-chain urokinase of mol wt 55,000 with a dissociation constant of 2.1 +/- 1.7 nmol/L, with 2.9 +/- 2.9 x 10(5) sites/cell were also identified. Single-chain urokinase of mol wt 55,000, but not the two-chain degradation product of mol wt 33,000 bound to the cells, implicating the amino-terminal aspects of the ligand in receptor recognition. When endothelial cells were stimulated with thrombin, an agent that modulates their fibrinolytic potential, both receptor types were modestly affected; urokinase binding increased 17%, whereas plasminogen binding decreased 19%. The presence and modulation of plasminogen and urokinase receptors provide a potentially important additional mechanism by which endothelial cells may regulate fibrinolysis.

Aminocaproic Acid↗
PubMed1988

Evidence that calpains and elastase do not produce the von Willebrand factor fragments present in normal plasma and IIA von Willebrand disease.

Recent evidence suggests that proteolysis plays an important role in some forms of inherited and acquired von Willebrand disease (vWD). Because calpains and one or more enzymes released from polymorphonuclear leukocytes are known to proteolyze von Willebrand factor (vWF) in vitro with resultant loss of large multimers similar to that seen in IIA vWD, they have been suggested as being responsible for the proteolysis in vivo. Using monoclonal epitope mapping, we have examined the proteolysis of the vWF subunit by porcine calcium-activated neutral proteases (calpains) and human leukocyte elastase to determine whether they produce the vWF proteolytic cleavage products seen in normal individuals and IIA vWD. Purified vWF was digested with porcine calpains I and II. We found no difference in the size, location, and quantity of the fragments produced by calpain I v calpain II. New fragments were detected of approximately 200, 170, 150, and 125 Kd. There was no evidence for generation of the native 140 and 176 Kd fragments. Some loss of the native fragments was seen, which suggests that they were further cleaved. Epitope mapping of the 170- and 150-Kd calpain-cleaved fragments revealed them to be from different parts of the molecule than the regions from which the native 176- and 140-Kd fragments derived. This was further supported by determination of the amino-terminal sequence of the calpain-cleaved 170- and 150-Kd fragments. Digestion of vWF with human leukocyte elastase produced new fragments at 210/205, 190, 170/165, 145/140, and 130/125 Kd. No generation of native fragments was detected. Monoclonal epitope mapping of the 145/140-Kd elastase-cleaved band proved that it derived from the carboxyl-terminal portion of the vWF molecule, whereas the native 140-Kd fragment is derived from the amino-terminal end. Neither calpains nor human leukocyte elastase produced the proteolyzed fragments present in normal and IIA vWD and, therefore, probably do not cause the loss of large multimers that is seen in that disorder.

Amino Acid Sequence↗
PubMed1987

Receptor mediated binding of the fibrinolytic components, plasminogen and urokinase, to peripheral blood cells.

Glu-plasminogen binds to platelets; the monocytoid line, U937, and the human fetal fibroblast line, GM1380 bind both plasminogen and its activator, urokinase. This study assesses the interaction of these fibrinolytic proteins with circulating human blood cells. Plasminogen bound minimally to red cells but bound saturably and reversibly to monocytes, granulocytes and lymphocytes with apparent Kd values of 0.9-1.4 microM. The interactions were of high capacity with 1.6 to 49 X 10(5) sites/cell and involved the lysine binding sites of plasminogen. Both T cells and non-rosetting lymphocytes and two B cell lines saturably bound plasminogen. Urokinase bound saturably to granulocytes, monocytes, non-rosetting lymphocytes and a B cell line, but minimally to T cells, platelets and red cells. Therefore, plasminogen binding sites of high capacity, of similar affinities, and with common recognition specificities are expressed by many peripheral blood cells. Urokinase receptors are also widely distributed, but less so than plasminogen binding sites. The binding of plasminogen and/or urokinase to these cells may lead to generation of cell-associated proteolytic activity which contributes to a variety of cellular functions.

Blood Cells↗
PubMed1987

Immunochemical and amino-terminal sequence comparison of two cytoadhesins indicates they contain similar or identical beta subunits and distinct alpha subunits.

Platelet membrane glycoprotein (GP) IIb-IIIa is functionally and antigenically related to proteins present on many cell types, suggesting that it is a member of the proposed cytoadhesin family of membrane proteins. We have compared the purified tissue vitronectin receptor (VnR) with GP IIb-IIIa. Anti-VnR immunoprecipitated GP IIb-IIIa and a related endothelial cell protein. In immunoblots, GP IIIa reacted with anti-VnR and the beta subunit of the VnR reacted with poly and monoclonal anti-GP IIIa. In contrast, the alpha subunit of the VnR failed to react either with a polyclonal anti-GP IIb or with monoclonal anti-GP IIb. Furthermore, the amino-terminal sequence of GP IIIa and the beta subunit of VnR were identical at determined residues while the alpha subunit and the GP IIb were different, but showed 33% identity. These data indicate the identity or close homology of GP IIIa and the beta subunit of the VnR. In contrast, the alpha subunit and GP IIb are distinct polypeptides which may be homologous. Since GP IIb-IIIa and the VnR differ in ligand recognition specificity, the data also suggest that this specificity may be governed by the alpha subunit of cytoadhesins.

Amino Acid Sequence↗
PubMed1987

Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets.

Synthetic peptides corresponding to the carboxyl terminus of the fibrinogen gamma chain inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to platelets, yet the active decapeptide sequence has only been found in fibrinogen to date. In contrast, all three proteins contain Arg-Gly-Asp sequences, and peptides containing Arg-Gly-Asp are potent inhibitors of their binding to activated platelets. We have analyzed the relationship between these peptide sets by direct binding assays. H12 (gamma 400-411) inhibited the binding of an Arg-Gly-Asp-containing peptide to platelets with similar dose response to inhibition of fibronectin binding. We have previously reported that GPIIb-IIIa binds to immobilized Arg-Gly-Asp peptides and can be eluted by Arg-Gly-Asp-containing peptides in solution. Both H12 and L10 (gamma 402-411) completely eluted GPIIb-IIIa bound to immobilized Arg-Gly-Asp peptides. Conversely, when GPIIb-IIIa was bound to immobilized L10, either L10 or an Arg-Gly-Asp peptide could elute it. Peptide specificity was established by the failure of Gly-Arg-Gly-Glu-Ser-Pro or acetylated L10 to elute GPIIb-IIIa from the immobilized peptides. These results indicate that the two peptide sets interact with the same receptor which contains GPIIb-IIIa.

Amino Acid Sequence↗
PubMed1987

Molecular cloning and chemical synthesis of a region of platelet glycoprotein IIb involved in adhesive function.

Membrane glycoprotein (GP) IIb-IIIa is a component of a platelet adhesive protein receptor. A region of the heavy chain of GPIIb, defined by the monoclonal antibody PMI-1, is involved in adhesion receptor function. We have localized and chemically synthesized this region of GPIIb. A cDNA clone that directs the synthesis of a fusion protein reactive with the PMI-1 antibody was isolated from a phage lambda gt11 expression library constructed with mRNA from an erythroleukemia (HEL) cell line. The deduced amino acid sequence of this clone indicates that it spans the light-heavy chain junction of GPIIb and contains a portion of the carboxyl terminus of the heavy chain and the amino terminus of the light chain. The PMI-1 epitope was found to be contained within a 9-kDa staphylococcal V8 protease fragment of GPIIb, and such a fragment was predicted within the putative heavy-chain sequence. A computerized antigen prediction program identified a single sequence with a high probability of containing a continuous epitope. A synthetic 17-residue peptide containing this sequence binds PMI-1 and inhibits PMI-1 binding to GPIIb-IIIa. The peptide-antibody complex has an approximate Kd of 1.2 microM, which compares to a Kd of 0.95 microM for PMI-1 binding to GPIIb. The region containing the PMI-1 epitope shows no similarity to corresponding regions of two other adhesion receptors, indicating that this portion of GPIIb may function in activities unique to the platelet receptor.

Amino Acid Sequence↗
PubMed1987

Epitope mapping of the von Willebrand factor subunit distinguishes fragments present in normal and type IIA von Willebrand disease from those generated by plasmin.

A small but consistent proportion of the von Willebrand factor (vWF) in normal plasma is composed of 189, 176, and 140 kD fragments cleaved from the 225 kD subunit. A monoclonal antibody map of vWF, based on the reactivity of individual antibodies with cyanogen bromide and tryptic fragments of known carboxy and/or amino termini, showed that in normal and IIA von Willebrand disease (vWD) plasmas the 140 kD fragment was derived from the amino-terminal region, whereas the 176 kD fragment was derived from the carboxy-terminal region of the subunit. In type IIA vWD, however, the fragments comprised a greater proportion of circulating vWF. In contrast, plasmin cleaved a 176 kD fragment from the amino terminus and a 145 kD fragment from the carboxy terminus of the subunit. Species similar to these plasmin-cleaved fragments were demonstrated in plasmas from four patients treated with fibrinolytic agents, but not in IIA vWD.

Antibodies, Monoclonal↗