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Biomedical subjects

T K Gartner

Publications and source records attributed to T K Gartner.

At least 19 recordsLinked to original sources
PubMed2001

Distinct domains of alphaIIbbeta3 support different aspects of outside-in signal transduction and platelet activation induced by LSARLAF, an alphaIIbbeta3 interacting peptide.

The peptide LSARLAF causes alphaIIbeta3-dependent platelet activation exemplified by secretion, aggregation, spreading and adhesion on fibrinogen, and tyrosine phosphorylation. alphaIIIbeta3-dependent outside-in signal transduction induced by LSARLAF was investigated in variant thrombasthenic platelets which lack most of the cytoplasmic domain of the integrin beta3 subunit (alphaIIbbeta3 delta724). These studies revealed that only certain aspects of this alphaIIbbeta3-dependent outside-in signaling were affected by the beta3 truncation. Specifically, alphaIIbbeta3 delta724 supported LSARLAF-induced platelet aggregation, agglutination and secretion, but failed to trigger cytoskeletal reorganization and platelet spreading on fibrinogen, despite the fact that PMA-induced non alphaIIbbeta3 mediated signaling caused spreading of these platelets on fibrinogen. Thus, distinct domains of alphaIIbbeta3 are required to support different aspects of LSARLAF-induced platelet activation. Furthermore, these studies suggest that not all alphaIIbbeta3-dependent platelet responses require an intact beta3 cytoplasmic tail.

Adenosine Diphosphate↗
PubMed2000

A naturally occurring mutation near the amino terminus of alphaIIb defines a new region involved in ligand binding to alphaIIbbeta3.

Decreased expression of functional alphaIIbbeta3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of alphaIIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P(145)A substitution, whereas a Chinese patient was doubly heterozygous for a P(145)L substitution. The mutations affect expression levels of surface alphaIIbbeta3 receptors on their platelets, which was confirmed by co-transfection of alphaIIb(P145A) and beta3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of alphaIIbbeta3 on affected platelets to interact with ligands. Moreover, when alphaIIb(P145A) and beta3 were stably coexpressed in Chinese hamster ovary cells, alphaIIbbeta3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after alphaIIbbeta3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to alphaIIb, induced PF4 secretion, indicating that the mutant alphaIIbbeta3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving alphaIIb(P145 )impair surface expression of alphaIIbbeta3 and that the alphaIIb(P145A) mutation abrogates ligand binding to the activated integrin. A comparative analysis of other alphaIIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alphaIIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)

Adolescent↗
PubMed2000

Evidence for new endothelial cell binding sites on fibrinogen.

In vitro assays were used to characterize adhesion of human aortic, microvascular and umbilical vein endothelial cells to various forms of immobilized fibrinogen. All three types of endothelial cells adhered to fibrinogen in a manner that was independent of the Aalpha-chain 572-574 RGD cell binding site. In fact, all three adhered to a fragment of the molecule which is composed of only one D domain (D1) of fibrinogen. A time course study revealed that extensive adhesion of endothelial cells on the ligand coated surface occurred between one and two hours incubation. The anti-fibrinogen gammaA-chain monoclonal antibody 4A5 as well as 4A5 Fabs, blocked adhesion of endothelial cells to fibrinogen, not vitronectin. The inhibitory effects of 4A5 seemed to be indirect because the endothelial cells adhered to the recombinant fibrinogen gamma407 (which lacks the gamma-chain AGDV sequence of the carboxyl terminal 4A5 binding site) as well as they did to normal recombinant fibrinogen. A recombinant fibrinogen lacking the gamma-chain AGDV sequence, containing RGE in place of RGD at the gamma-chain 572-574 and 95-97 positions, also supported endothelial cell adhesion. The anti-alphavbeta3 antibody, LM609, blocked adhesion of endothelial cells to fibrinogen. The peptide GRGDSP inhibited endothelial cell adhesion on fibrinogen and vitronectin. These results demonstrate that alphavbeta3 mediated adhesion (attachment and spreading) of HUVECs to fibrinogen may use a site in the D domain of fibrinogen and is not dependent on the Aalpha-chain RGD (95-97 and 572-574) sequences, as has been shown in shorter term (where cells were rounded) experiments, or the alphaA-chain 408-411 cell binding sites. Thus, the data reveal the existence of another unidentified site(s) on fibrinogen which can support the irreversible adhesion (attachment and spreading) of endothelial cells.

Binding Sites↗
PubMed1998

Peptide LSARLAF activates alpha(IIb)beta3 on resting platelets and causes resting platelet aggregate formation without platelet shape change.

Adhesion of resting platelets to fibrinogen was enhanced by a peptide which was designed to bind near the presumptive fibrinogen gamma-chain binding site of the alpha subunit of the integrin alpha(IIb)beta3. This peptide, but not a scrambled control peptide, induced adhesion of resting platelets to fibronectin, vitronectin, von Willebrand factor, and monovalent (lacks one functional gamma-chain) fibrinogen. Resting platelets not treated with the agonist peptide did not adhere to these ligands. Agonist peptide induced adhesion of resting platelets to Fg was not secretion dependent and was inhibited by the monoclonal antibody 7E3. The agonist peptide caused aggregation of resting platelets on resting platelets adherent to immobilized Fg without causing platelet shape change. Therefore, the agonist peptide may activate alpha(IIb)beta3 by directly inducing a conformation change in the receptor on resting platelets.

Adenosine Diphosphate↗
PubMed1997

The role of putative fibrinogen Aalpha-, Bbeta-, and GammaA-chain integrin binding sites in endothelial cell-mediated clot retraction.

In this study, endothelial cell-mediated clot retraction was supported by fibrin generated from several purified fractions of plasma fibrinogen, purified proteolytic fragments of plasma fibrinogen, recombinant normal fibrinogen, and recombinant variant fibrinogen. These results were surprising because some of these fibrinogens lack domains that are known binding sites for the integrin receptors that support clot retraction. Specifically, fibrinogens lacking Aalpha-chain RGD residues at 572-574 or lacking the gamma-chain residues AGDV 408-411 supported endothelial cell-mediated clot retraction as well as intact fibrinogen. Thus, clot retraction mediated by endothelial cells is not dependent on either of these sites. A variety of monoclonal antibodies against the integrin alphavbeta3 partially inhibited the endothelial cell-mediated retraction of clots formed from plasma fibrinogen. As expected, an antibody to the platelet integrin alphaIIbbeta3 did not inhibit endothelial cell-mediated clot retraction. These results indicate that this retraction is mediated at least in part by alphavbeta3. These results support the conclusion that (a) neither of the two fibrinogen cell binding sites described above is required to support clot retraction or that (b) either site alone or in conjunction with other fibrin(ogen) region(s) can support clot retraction. Thus, endothelial cell-mediated clot retraction appears to be dependent on fibrinogen cell binding sites other than those required to support adhesion of resting platelets to immobilized fibrinogen and platelet aggregation.

Binding Sites↗
PubMed1997

The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin alphaIIbbeta3.

A novel peptide (designed to bind to alphaIIbbeta3) caused platelet aggregation and aggregation-independent secretion of the contents of alpha-granules in an alphaIIbbeta3-dependent fashion. The agonist peptide induced secretion in the presence of prostaglandin E1. In cell-free assays, alphaIIbbeta3 bound specifically to immobilized agonist peptide and the peptide enhanced the binding of fibrinogen to immobilized alphaIIbbeta3. The agonist peptide apparently activates alphaIIbbeta3 by directly inducing a conformational change in the receptor. This change activates the platelets and causes secretion in a manner independent of fibrinogen binding.

Animals↗
PubMed1996

Variabilin, a novel RGD-containing antagonist of glycoprotein IIb-IIIa and platelet aggregation inhibitor from the hard tick Dermacentor variabilis.

A novel inhibitor of human platelet aggregation, named variabilin, was isolated from salivary glands of the hard tick Dermacentor variabilis using a combination of gel filtration and high pressure liquid chromatography. Variabilin was a potent antagonist of the fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa; alphaIIbbeta3) and the vitronectin receptor alphavbeta3. Amino acid sequence analysis by Edman degradation revealed that it has 47 residues, with a molecular weight of 4968.5. Like many other naturally occurring antagonists of GPIIb-IIIa, variabilin contains the RGD (Arg-Gly-Asp) motif. However, unlike the RGD-containing antagonists of GPIIb-IIIa, the RGD sequence of variabilin is not positioned in a loop bracketed by cysteine residues. It has little sequence homology to the other known naturally occurring antagonists of GPIIb-IIIa, including the disintegrins from snakes, decorsin and ornatin from leeches, and disagregin from soft ticks. Variabilin is the first RGD-containing antagonist isolated from ticks.

Amino Acid Sequence↗
PubMed1995

Fibronectin peptide DRVPHSRNSIT and fibronectin receptor peptide DLYYLMDL arrest gastrulation of Rana pipiens.

Gastrulation is characterized by dramatic cell migration which is thought to require the interaction of cell adhesion molecules with extracellular molecules. We have tested two novel peptides, a fibronectin peptide and a fibronectin receptor peptide, for their effects on gastrulation of the leopard frog Rana pipiens. The fibronectin peptide DRVPHSRNSIT corresponds to residues 1373-1383 of the cell-binding domain of fibronectin; the receptor peptide DLYYLMDL corresponds to residues 124-131 of beta 1 subunit of a variety of integrins including alpha 5 beta 1. Either of these peptides significantly inhibited gastrulation after being microinjected into mid-blastulae. These results indicate that these sequences may correspond to the ligand/receptor interaction sites of fibronectin and its receptor(s).

Amino Acid Sequence↗
PubMed1994

Complementary peptides that interfere with platelet aggregation and adherence.

This article describes the application of the molecular recognition hypothesis to the critically important process of fibrinogen binding to platelets, a process that is the subject of extensive and intensive basic and clinical research. The objectives of the studies summarized below were to design, synthesize, and characterize peptides that can inhibit the binding of fibrinogen and related ligands to human platelets and thereby prevent platelet aggregation, adhesion, and clot retraction. The purpose of doing this work was twofold: first, to determine whether the molecular recognition hypothesis could serve as a useful rationale for the design of peptides that can specifically inhibit the binding of fibrinogen and related ligands to platelets; and second, to use these peptides to try to learn where fibrinogen binds to the platelet fibrinogen receptor. It was hoped that the results obtained not only would provide insight into platelet function but also might provide a rationale for the design of a clinically useful anti-thrombotic agent. Although our studies are not complete, they have resulted in the design of a variety of peptides that can inhibit platelet aggregation, adhesion, and clot retraction as a consequence of specifically inhibiting the binding of fibrinogen and related ligands to the platelet fibrinogen receptor. Although none of these peptides appears to be ligand specific, one or two of them may be specific for platelets.

Amino Acid Sequence↗
PubMed1994

Characterization of adhesion of non-exogenously stimulated and resting platelets in normal plasma to fibrinogen and its fragments.

Platelet adhesion to various forms of fibrinogen was studied using platelets in plasma and washed platelets. The study was designed to determine if platelets prepared with minimal handling in plasma at physiological pH containing normal levels of Ca2+ have different requirements for adhesion to immobilized fibrinogen than do washed platelets tested in the absence of plasma. Exposure of platelets to citrate and low pH did not seem to affect the requirements of washed platelets for adhesion to fibrinogen. Nonetheless, behavioural differences between these two types of platelets were seen. Surprisingly, in the absence of exogenous activation normal platelets in plasma behaved qualitatively as stimulated washed platelets. That is, both types of platelets adhered to all forms of fibrinogen which possessed at least one gamma-chain carboxyl terminal platelet binding site. Platelets in plasma treated with prostaglandin E1 (resting platelets) adhered only to forms of fibrinogen which contained two gamma-chain platelet binding sites. These observations also demonstrate that the fibrinogen alpha-chain arginine-glycine-aspartic acid-phenylalanine and arginine-glycine-aspartic acid-serine sequences are not necessary or sufficient to mediate the adhesion of resting or stimulated platelets in plasma to fibrinogen. The presence of endogenous adenosine diphosphate appears to account, at least in part, for the ability of normal platelets in plasma to adhere to forms of fibrinogen which have only one gamma-chain platelet binding site.

Adenosine Diphosphate↗
PubMed1994

Antibodies to GPIIb alpha (300-312) inhibit Fg binding, clot retraction, and platelet adhesion to multiple ligands.

We previously reported that a peptide with the sequence Gly-Ala-Pro-Leu (GAPL) found as residues 309-312 in glycoprotein IIb alpha (GPIIb) comprises at least part of a Fg binding site on GPIIb (1). Subsequent studies demonstrated that a peptide corresponding to residues 300-312 of GPIIb alpha can bind to Fg and Vn, and is a potent inhibitor of platelet aggregation and the adhesion of activated platelets to at least four adhesive ligands: Fg, Fn, Vn, and vWf (2). Here, the production and initial characterization of polyclonal antibodies against this peptide are described. ELISAs and dot-blot assays reveal the specificity of the antibodies for the peptide immunogen. In immunoblots, the antibodies recognize GPIIb under reducing conditions. The binding of Fg to immobilized GPIIb/IIIa, the rate of clot retraction and the adhesion of stimulated platelets to Fg, fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWf) were inhibited by these antibodies, but not by a control IgG. These results together with our earlier published data indicate that GPIIb alpha (300-312) comprises at least part of a common ligand binding site within the integrin alpha IIb subunit.

Amino Acid Sequence↗
PubMed1993

Characterization of adhesion of "resting" and stimulated platelets to fibrinogen and its fragments.

Adhesion of resting and stimulated platelets to immobilized fibrinogen (Fg) was characterized using various forms of Fg, receptor peptide mimics, and antibodies to glycoprotein (GP) IIb/IIIa and Fg. Resting platelets adhered to Fg, but to less than half the extent of the same platelets stimulated with epinephrine/ADP. The adhesion of resting and stimulated platelets to Fg was inhibited by a receptor peptide mimic (G13, a peptide corresponding to residues 300-312 of GPIIb), anti-GPIIb/IIIa antibodies, and a monoclonal antibody (4A5) against the carboxyl terminus of the gamma chain of Fg. The results presented here demonstrate that the alpha chain RGD platelet recognition sites are not required to mediate the adhesion of either stimulated or resting platelets to immobilized Fg. Although stimulated platelets can adhere extensively to monomeric Fg containing one functional gamma chain, resting platelets require bivalent Fg containing two functional gamma chains to mediate irreversible adhesion to Fg.

Amino Acid Sequence↗
PubMed1992

A peptide corresponding to GPIIb alpha 300-312, a presumptive fibrinogen gamma-chain binding site on the platelet integrin GPIIb/IIIa, inhibits the adhesion of platelets to at least four adhesive ligands.

The platelet fibrinogen (Fg) receptor (GPIIb/IIIa) is an integrin which plays a critical role in hemostasis by recognizing at least the four adhesive ligands: Fg, fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWf). We reported that residues 309-312 of GPIIb alpha appear to comprise at least part of a Fg binding site on the Fg receptor (Gartner, T. K., and Taylor, D. B. (1990) Thromb. Res. 60, 291-309). Here we report that the peptide GPIIb alpha 300-312 (G13) inhibits platelet aggregation and binds Fg and Vn. Significantly, this peptide inhibits the adhesion of stimulated platelets to Fg, Fn, Vn, and vWf, but not the adhesion of resting platelets to Fn. Thus, GPIIb 300-312 may constitute a specific but common recognition site on GPIIb/IIIa for both LGGAKQAGDV- and RGD-containing ligands.

Amino Acid Sequence↗
PubMed1991

The peptides APLHK, EHIPA and GAPL are hydropathically equivalent peptide mimics of a fibrinogen binding domain of glycoprotein IIb/IIIa.

The anticomplementarity hypothesis predicted that the peptides APLHK, EHIPA, GAPL and LGVPPR would be functional mimics of a fibrinogen binding domain(s) in the fibrinogen receptor. The peptides APLHK and EHIPA were derived by translation of the cRNA of vitronectin m-RNA. The peptides GAPL and LGVPPR result from translations of the cRNA of von Willebrand factor m-RNA. The peptides APLHK, EHIPA, and GAPL, but not LGVPPRT, are hydropathically equivalent and inhibit fibrinogen binding to platelets. APLHK and EHIPA are hydropathic retromers. Thus for one pair of these peptides, the direction of their backbones did not affect function.

Amino Acid Sequence↗
PubMed1991

External Na+ is not required for Ca2+ mobilization in platelets stimulated with rattlesnake lectin or alpha-thrombin.

The effects of extracellular sodium on platelet aggregation and calcium mobilization in platelets stimulated with either rattlesnake (Crotalus atrox) lectin (RSL) or alpha-thrombin were compared. The absence of extracellular sodium had no effect on platelet aggregation or calcium mobilization in response to all levels of RSL tested. In contrast platelet aggregation was sodium-dependent in response to less than or equal to .2 units/ml alpha-thrombin. Surprisingly, calcium mobilization occurred in platelets treated with a threshold level of alpha-thrombin in the absence of external sodium. Thus sodium-dependent platelet aggregation in response to a low dose of thrombin apparently is not the result of sodium-dependent calcium mobilization.

Blood Platelets↗
PubMed1991

The peptide Glu-His-Ile-Pro-Ala binds fibrinogen and inhibits platelet aggregation and adhesion to fibrinogen and vitronectin.

Antiplatelet agents are clinically useful as antithrombotic entities. The importance of antiplatelet agents led us to design, synthesize, and characterize a new antiplatelet peptide. This peptide is a presumptive mimic of a ligand binding site on the platelet fibrinogen receptor. Unlike peptides related to Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val that bind to the fibrinogen receptor, this peptide binds to fibrinogen. The anticomplementarity hypothesis was used to design this presumptive peptide mimic of the vitronectin binding site on the fibrinogen receptor, glycoprotein IIb/IIIa complexes. The resulting peptide (Glu-His-Ile-Pro-Ala) has the characteristics of a fibrinogen binding site mimic: It binds fibrinogen and inhibits both the adhesion of platelets to fibrinogen and platelet aggregation. The peptide also inhibits the adhesion of platelets to vitronectin. The antiplatelet activity of this mimic peptide was dependent on its amino acid sequence, since closely related analogues were either inactive or less active inhibitors of platelet function than the original peptide. These results demonstrate that the peptide Glu-His-Ile-Pro-Ala has the characteristics expected of a mimic of a glycoprotein IIb/IIIa ligand binding site.

Adenosine Diphosphate↗
PubMed1990

The amino acid sequence Gly-Ala-Pro-Leu appears to be a fibrinogen binding site in the platelet integrin, glycoprotein IIb.

The amino acid sequence Gly-Ala-Pro-Leu-Arg-Val is predicted by the anticomplementarity hypothesis to be a fibrinogen binding site on human platelet fibrinogen receptors. The peptide Ala-Pro-Leu-Arg-Val binds fibrinogen and inhibits platelet aggregation and clot retraction. The peptide Gly-Ala-Pro-Leu is the shortest sequence within the predicted sequence which potently inhibits the adhesion of platelets to fibrinogen and platelet aggregation. The sequence Gly-Ala-Pro-Leu is present as residues 309-312 in glycoprotein IIb, the alpha-subunit of the glycoprotein IIb/IIIa complex, the fibrinogen receptor. The sequence Gly-Ala-Pro-Leu is present in 4 of 8 integrin alpha-subunits and Gly-Ala-Pro is present in 8 of 8 integrin alpha-subunits.

Amino Acid Sequence↗