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S Timmons

Publications and source records attributed to S Timmons.

At least 37 records · Page 2Linked to original sources
PubMed1989

Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis.

We have previously shown that the alpha chain of human fibrinogen interacts directly with ADP-activated human platelets [Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., & Doolittle, R. F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2068]. Now, we report that platelet receptor recognition domains are localized on two CNBr fragments of the human fibrinogen alpha chain. They encompass residues 92-147 and 518-584, which inhibit 125I-fibrinogen binding to ADP-stimulated platelets. The inhibitory CNBr fragment alpha 92-147 contains the RGD sequence at residues 95-97. Synthetic peptides encompassing this sequence were inhibitory while peptide 99-113 lacking the RGD sequence was inactive. The synthetic peptide RGDF, corresponding to residues alpha 95-98, inhibited the binding of 125I-fibrinogen to ADP-treated platelets (IC50 = 2 microM). However, the peptides containing sequence RGDF, with residues preceding Arg95 or following Phe98, were less inhibitory. It appears that the sequence alpha 95-98 constitutes a platelet receptor recognition domain which is constrained by flanking residues. The second inhibitory CNBr fragment, alpha 518-584, also contains the sequence RGD at positions 572-574. Synthetic peptides overlapping this sequence were inhibitory, while peptides lacking the sequence RGDS were not reactive. Thus, another platelet reactive site on the alpha chain encompasses residues 572-575 containing sequence RGDS. In conclusion, the platelet receptor recognition domains on the human fibrinogen alpha chain in the amino-terminal and in the carboxy-terminal zones contain the ubiquitous cell recognition sequence RGD shared with other known adhesive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Platelet receptor recognition domain on the gamma chain of human fibrinogen and its synthetic peptide analogues.

We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767]. To study the correlation between the structure of this segment of the gamma chain and its reactivity toward receptors on ADP-activated human platelets, we designed a series of analogues containing replacements at 9 out of 12 positions. A double substitution of the normal His400-His401 sequence by Ala-Ala reduced the inhibitory potency of the dodecapeptide 3-fold. When Lys406 was replaced by Arg, the inhibitory potency of the dodecapeptide decreased 15 times. On the other hand, substitution of Ala408 with Arg increased the inhibitory potency of the dodecapeptide 6-fold. A drastic decrease in the reactivity of the dodecapeptide toward platelet receptors was observed when Val411 was replaced by leucine or cysteine or tyrosine. A 3-fold decrease in reactivity was noted when Val411 was substituted with phenylalanine. Amidation of the carboxy-terminal Val411 also produced a significant decrease in dodecapeptide reactivity. With seven residues (His400, His401, Leu402, Lys406, Gln407, Asp410, and Val411) preserved, substitution of the intervening five amino acids with nonpolar leucine or polar serine, increasing or decreasing the hydrophobicity of the dodecapeptide, reduced more than 16-fold its inhibitory potency. Rabbit antibody Fab fragments directed against the human fibrinogen gamma-chain peptide encompassing residues 385-411 inhibited 50% of 125I-fibrinogen binding at a 2:1 stoichiometry with regard to 125I-fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Antiplatelet "hybrid" peptides analogous to receptor recognition domains on gamma and alpha chains of human fibrinogen.

Platelet receptor recognition domains are located on the gamma and alpha chains of human fibrinogen. The former encompasses residues 400-411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767], and the latter is present in two loci on the alpha chain (alpha 95-97 and alpha 572-574) [Hawiger, J., Kloczewiak, M., Bednarek, M. A., & Timmons, S. (1989) Biochemistry (first of three papers in this issue)]. Peptide gamma 400-411 (HHLGGAKQAGDV) inhibited aggregation of ADP-treated platelets mediated not only by gamma-chain but also by alpha-chain multimers. Peptide alpha 572-575 (RGDS) inhibited aggregation of platelets mediated by alpha-chain as well as gamma-chain multimers. These results indicate that the platelet receptor for fibrinogen is isospecific with regard to the domain present on alpha and gamma chains. Subsequent "checkerboard" analysis of combinations of gamma 400-411 and alpha 572-575 showed that the inhibitory effect toward binding of 125I-fibrinogen was additive rather than synergistic. Next, a series of "hybrid" peptides was constructed in which the alpha-chain sequence RGDF (alpha 95-98) replaced the carboxy-terminal segment of gamma 408-411. The dodecapeptide HHLGGAKQRGDF was inhibitory with concentration, causing 50% inhibition of binding (IC50) at 6 microM, 5 times more potent than gamma 400-411. The shorter peptides AKQRGDF and KQRGDF were also more inhibitory than gamma 400-411. The second series of hybrid peptides was constructed with the alpha-chain sequence RGDS preceding the sequence of gamma 400-411 or sequence RGDV following it.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1988

Plasma components are required for platelet activation by the toxin of Loxosceles reclusa.

We have used a partially purified toxin from the venom of the brown recluse spider, Loxosceles reclusa, to study its effects on human platelets isolated from plasma proteins. This toxin, which produced skin necrosis in rabbits, contained sphingomyelinase D activity. The toxin induced platelet aggregation and secretion of [3H]serotonin in human plasma but not in buffer or in human neonate plasma. Ca2+ was required for the interaction of toxin, platelets, and plasma factor(s). The addition of C-reactive protein restored aggregation and serotonin release of platelets incubated in human neonate plasma. The ADP-degrading enzyme, apyrase, and the non-steroidal, anti-inflammatory drug, indomethacin, inhibited platelet aggregation, suggesting that ADP secreted from platelet storage granules and indomethacin-sensitive pathway(s) are involve in the toxin-induced human platelet activation (aggregation and serotonin release). Generation of platelet activating factor (PAF) from the platelet by brown recluse toxin is not likely since the PAF receptor antagonist, BN 52021, did not inhibit platelet aggregation induced by the brown recluse toxin.

Adenosine Diphosphate↗
PubMed1988

Modulation of human platelet protein kinase C by endotoxic lipid A.

Lipid A is the toxic principle of lipopolysaccharide of gram-negative bacteria, which causes a spectrum of changes in blood cells and vascular cells. We now report that human platelets are directly stimulated by endotoxic lipid A that activates protein kinase C. Rapid phosphorylation of a human platelet protein of Mr 47,000, a marker of protein kinase C activation, accompanies secretion of [14C]serotonin and aggregation triggered by endotoxic lipid A. These events are time and concentration dependent, with phosphorylation reaching maximum in 2 min and the concentration of lipid A causing a 50% effect (EC50) between 12 and 15 microM. Phospholipase C activation in lipid A-stimulated platelets was not observed as judged by a lack of generation of [3H]diacylglycerol in [3H]arachidonic acid-labeled platelets and a lack of generation of [32P]-phosphatidic acid in 32PO4-labeled platelets. Lipid A did not induce formation of TXA2 as measured by radioimmunoassay for TXB2. The stimulation of human platelets and activation of protein kinase C by endotoxic lipid A was blocked by lipid X, a structural precursor of lipid A. Lipid X also blocked the stimulation of human platelets by phorbol 12-myristate 13-acetate, suggesting that lipid A, lipid X and phorbol ester share reactive site(s) on the human platelet membrane. Although lipid X inhibited thrombin-induced phosphorylation of P47 it did not suppress secretion of [14C]serotonin, indicating the role of protein kinase C-independent pathways in platelet stimulation by thrombin. The inhibitory effect of lipid X did not involve generation of cyclic AMP in human platelet membrane preparations. These results indicate that human platelets are stimulated by endotoxic lipid A, a naturally occurring biologic modifier of protein kinase C. Due to the widespread presence of this enzyme in blood cells, vascular cells, and neurons, its modulation by lipid A may represent a significant mechanism underlying hematologic and circulatory derangements observed in endotoxic shock in humans.

Blood Platelets↗
PubMed1987

Reactivity of chemically cross-linked fibrinogen and its fragments D toward the staphylococcal clumping receptor.

It has been established that the binding domain for the staphylococcal clumping receptor exists in fragment D of human fibrinogen [Hawiger J., Timmons, S., Strong, D. D., Cottrell, B. A., Riley, M., & Doolittle, R. F. (1982) Biochemistry 21, 1407; Strong, D. D., Laudano, A., Hawiger, J., & Doolittle, R. F. (1982) Biochemistry 21, 1414]. To examine the role of valency in the adhesive function of fibrinogen, its fragments were prepared by digestion with plasmin in the presence of calcium and purified by a two-step chromatographic procedure. Fragments D1 and E did not induce the staphylococcal clumping reaction. After they were prepared in oligomeric form by chemical cross-linking with glutaraldehyde, fragment D1 (Mr 94,000) became functionally reactive toward the staphylococcal clumping receptor, and fragment D3 (Mr 75,000) and fragment E (Mr 50,000) remained inactive. Fragment D dimer derived from enzymatic cross-linking was not reactive. Human fibrinogen cross-linked with glutaraldehyde usually reached a 250 times higher reactivity toward the staphylococcal clumping receptor, depending on the condition of the cross-linking reaction. It is concluded that the valency of fibrinogen in regard to its receptor binding domain and the availability of this domain are essential for the staphylococcal clumping reaction.

Aldehydes↗
PubMed1986

Mechanism of human platelet activation by endotoxic glycolipid-bearing mutant Re595 of Salmonella minnesota.

The mechanism through which human blood platelets interact with gram-negative bacteria with well-defined structural variations in endotoxic lipopolysaccharide was studied. Secretion of 14C-serotonin and aggregation of platelets separated from plasma proteins were observed on challenge with rough mutant Re595 of Salmonella minnesota possessing a glycolipid outer layer composed of Lipid A and 2-keto-3-deoxyoctonate (KDO) but lacking heptose phosphate in the core and O-polysaccharide in its outer portion. Both 14C-serotonin secretion and platelet aggregation were concentration-dependent, with a half-maximum response at the ratio of one bacterial colony-forming unit (CFU) to two platelets. The aggregation of human platelets induced by mutant Re595 was divalent cation-dependent and required secretion of ADP and fibrinogen from platelet storage granules because it was inhibited by chelators, by the ADP-splitting enzyme apyrase, and by monospecific antifibrinogen Fab fragments. The synthetic peptide analog of the platelet receptor recognition site on the gamma chain of fibrinogen, gamma 400-411, inhibited platelet aggregation induced by mutant Re595 (IC50 160 mumol/L), whereas serotonin secretion was unaffected. Tetrapeptide, RGDS, analogous to human fibrinogen alpha chain (alpha 572-575) and to the cell adhesion site of fibronectin, also inhibited aggregation induced by mutant Re595 (IC50 60 mumol/L). Secretion of 14C-serotonin was preceded by a very rapid phosphorylation of a platelet protein of mol wt 47,000, which is associated with protein kinase C activation. Myosin light chain (mol wt 20,000) was also phosphorylated. Both phosphoproteins were dephosphorylated while secretion was reaching maximum. Furthermore, release of 3H-arachidonic acid from platelet phospholipids and generation of thromboxane B2 via the cyclooxygenase pathway were observed. Inhibition of this pathway with acetylsalicylic acid (10(-4) mol/L) or indomethacin (5 X 10(-4) mol/L) reduced 14C-serotonin secretion and platelet aggregation. The role of Lipid A in the interaction of mutant Re595 with human platelets was deduced from the inhibitory effect of the Lipid A-binding protein present in Limulus amebocyte lysate. Likewise, polymyxin B, known to complex with Lipid A, was inhibitory. The reactivity of mutant Re595 toward platelets was attenuated by mild acid hydrolysis, during which KDO was dissociated from the glycolipid, and by alkaline hydrolysis, which breaks ester-linked fatty acids in Lipid A. In contrast to mutant Re595, strain S218 of S minnesota bearing "complete" endotoxic lipopolysaccharide did not induce secretion and aggregation of human platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1984

Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the gamma chain.

Binding of fibrinogen to human platelets depends on the interaction of the gamma-chain carboxy-terminal segment with specific receptors exposed by different agonists such as ADP, epinephrine, and thrombin. The functions of a series of synthetic peptides encompassing the sequence of the 15 carboxy-terminal residues of the gamma chain were investigated in this study. Both pentadecapeptide (gamma 397-411) and dodecapeptide (gamma 400-411) inhibited binding of 125I-fibrinogen to ADP-treated platelets, with the concentration causing 50% inhibition (IC50) being 28 microM. In comparison, decapeptide (gamma 402-411) was almost 4 times less active (IC50 = 106 microM), thus suggesting that the two histidine residues (gamma 400-401) are required for a full inhibitory effect. A heptapeptide (gamma 405-411) had a similar effect (IC50 = 102 microM) whereas a pentapeptide (gamma 407-411) was even less inhibitory (IC50 = 190 microM), indicating that the lack of lysine (gamma 406) further diminishes the reactivity of the platelet recognition site on the gamma chain of human fibrinogen. The heptapeptide (gamma 400-406) containing two histidine residues and derived from the dodecapeptide by proteolytic degradation with trypsin had very low inhibitory activity. The synthetic peptides inhibited fibrinogen-supported platelet aggregation in the same order of decreasing reactivity: pentadecapeptide = dodecapeptide greater than decapeptide = heptapeptide greater than pentapeptide. Modified synthetic pentadecapeptides bearing tyrosine or cysteinyltyrosine at the amino terminal were prepared to provide a means for radiolabeling and for formation of molecules of higher valency.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

ADP-dependent common receptor mechanism for binding of von Willebrand factor and fibrinogen to human platelets.

Human von Willebrand factor (vWF) and fibrinogen are adhesive plasma glycoproteins essential for formation of a platelet hemostatic plug. We investigated the role of ADP and fibrinogen in binding of vWF to platelets in vitro. Binding of 125I-labeled vWF to human platelets separated from plasma proteins and treated with ADP was specific, and time and concentration dependent, reaching equilibrium at 20 min and approaching saturation at 12 micrograms/ml. The binding was inhibited by EDTA and by prostaglandin I2, a known activator of platelet adenylate cyclase. A purine nucleotide affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies the ADP binding sites on the human platelet membrane, prevented binding of vWF induced with ADP, as well as with human thrombin and with ionophore A23187, agents known to cause platelet ADP secretion. By comparison, FSBA did not inhibit binding of vWF induced by ristocetin, indicating that the ristocetin mechanism is not dependent on ADP. Human fibrinogen inhibited in a competitive manner the ADP-induced binding of 125I-labeled vWF (9 micrograms/ml) with an IC50 of 25 micrograms/ml. Conversely, unlabeled vWF inhibited ADP-induced binding of 125I-labeled fibrinogen (60 micrograms/ml) with an IC50 of 16 micrograms/ml. A synthetic dodecapeptide (Mr, 1188), analogous with the specific platelet receptor recognition site of human fibrinogen gamma chain (gamma 400-411), inhibited binding of both 125I-labeled vWF and 125I-labeled fibrinogen to ADP-treated platelets, whereas it was without effect on binding of 125I-labeled vWF to ristocetin-treated platelets. These data indicate that vWF and fibrinogen have a common receptor mechanism for their interaction with human platelets that is dependent on ADP occupancy of its binding sites and is recognized by the sequence of 12 amino acid residues at the carboxyl terminus of the human fibrinogen gamma chain.

Adenosine Diphosphate↗
PubMed1983

Interaction of fibrinogen with staphylococcal clumping factor and with platelets.

Fibrinogen, a clottable plasma glycoprotein, participates in cell adhesion phenomena involving prokaryotic cells, e.g. staphylococci, and eukaryotic cell fragments, e.g. platelets. Among the three chains (alpha, beta, gamma) of human fibrinogen, the gamma chain bears the main site recognizing the staphylococcal clumping receptor and human platelet receptor induced by ADP. The platelet receptors are also recognized, albeit less avidly, by a site associated with the alpha chain. The gamma chain site recognizing staphylococcal clumping factor exists on the COOH-terminal segment of this chain encompassing the 15 residues (gamma 397-411) including the COOH-terminal valine. The location of the gamma chain site interacting with the human platelet receptor had been pinpointed to the 27 residue CNBr COOH-terminal segment (gamma 385-411). The results of enzymatic degradation of the 27-residue peptide indicate that the continuity of the last 15 amino acid residues at the COOH-terminal end of the gamma chain of human fibrinogen seems to be essential for its interaction with human platelets. The sequence of the gamma chain interacting with the platelet receptor (gamma 385-411) indicates that this segment is a unique region of fibrinogen endowed with three important functions: cross-linking of fibrin, clumping of staphylococci, and aggregation of platelets. [Note added in proof: Recently we obtained evidence that dodecapeptide gamma 393-411 fully retains platelet receptor recognition site (Kloczewiak et al. 1983. Clin. Res 31:534A.)]

Amino Acid Sequence↗
PubMed1983

Recognition site for the platelet receptor is present on the 15-residue carboxy-terminal fragment of the gamma chain of human fibrinogen and is not involved in the fibrin polymerization reaction.

A pentadecapeptide, derived from a staphylococcal protease digest of the 27-residue carboxy-terminal cyanogen bromide fragment of human fibrinogen gamma chain, inhibits binding of 125I-fibrinogen to human platelet receptors and aggregation of platelets induced by ADP and fibrinogen. Amino acid composition and NH2 terminal analysis indicate that the isolated pentadecapeptide corresponds to residues 397 to 411 of the gamma chain. A synthetic peptide also inhibited binding of 125I-fibrinogen and aggregation of platelets. In contrast, the isolated pentadecapeptide and its parent 27-residue fragment lack inhibitory activity toward the polymerization reaction of fibrin monomer. Thus, the site recognizing the platelet receptor encompasses residues 397-411 of the gamma chain of fibrinogen and is distinct from the site(s) involved in polymerization of fibrin monomers.

Amino Acid Sequence↗
PubMed1982

gamma and alpha chains of human fibrinogen possess sites reactive with human platelet receptors.

Fibrinogen, a clottable plasma protein, agglutinates both prokaryotic cells (e.g., staphylococci) and eukaryotic cell fragments (e.g., platelets) through interaction with specific receptors. To identify the region of the fibrinogen molecule responsible for its interaction with human platelets, we prepared polypeptide chain subunits (alpha, beta, and gamma) of human fibrinogen by reduction and carboxymethylation. A mixture of the chains induced aggregation (clumping) of human platelets separated from plasma proteins and treated with ADP. When individual chains of fibrinogen were tested, gamma-chain multimers caused platelet aggregation at a molar concentration comparable with that of intact human fibrinogen. The beta chain remained inactive, and the alpha chain was 1/4th to 1/5th as reactive as the gamma chain. Monospecific antibody fragments against the gamma chain inhibited binding of 125I-labeled fibrinogen to the human platelet receptor and blocked aggregation of platelets induced by ADP in the presence of fibrinogen or gamma-chain multimers. These results indicate that the gamma chain of human fibrinogen bears the main site for interaction with the platelet receptor.

Antigen-Antibody Reactions↗
PubMed1980

Platelet aggregates in peripheral and coronary-sinus blood in patients with spontaneous coronary-artery spasm.

Five out of twenty patients with vasotonic angina had frequent spontaneous attacks with dramatic ST-segment alterations. These attacks were consistently associated with an increase in platelet aggregates in coronary-sinus blood but not arterial blood. This increase in platelet aggregates during coronary-artery spasm suggests a potential causal role for the platelet in this subset of patients with ischaemic heart-disease. However, the increase in platelet aggregates could be secondary to reduced coronary blood-flow.

Adult↗