Biomedical subjects

M Kloczewiak

Publications and source records attributed to M Kloczewiak.

At least 19 recordsLinked to original sources

PubMed1997

Effect of anticoagulants on binding and neutralization of lipopolysaccharide by the peptide immunoglobulin conjugate CAP18(106-138)-immunoglobulin G in whole blood.

The 18-kDa cationic protein CAP18 is an antimicrobial protein isolated from rabbit granulocytes that binds lipopolysaccharide (LPS) and inhibits many of its biological activities. We covalently coupled a synthetic peptide representing amino acids 106 to 138 of CAP18 to human immunoglobulin G (IgG) by using the heterobifunctional linker N-succinimidyl-3-(2-pyridyidithio)propionate. The ability of CAP18(106-138)-IgG to bind and neutralize LPS in whole blood in the presence and absence of anticoagulants was studied. Both CAP18(106-138) and CAP18(106-138)-IgG significantly suppressed LPS-induced tumor necrosis factor (TNF) production in whole blood in the absence of anticoagulants. EDTA potentiated the ability of CAP18(106-138) and CAP18(106-138)-IgG to decrease LPS-induced TNF production in a dose-dependent manner. In contrast, heparin inhibited the ability of CAP18(106-138) and CAP18(106-138)-IgG to suppress LPS-induced TNF production. EDTA also enhanced LPS capture in a fluid-phase binding assay that utilizes magnetic anti-IgG beads to capture CAP18(106-138)-IgG (and bound [3H]LPS) in whole blood. In contrast, heparin inhibited the binding dose dependently. We conclude that CAP18(106-138)-IgG binds to and neutralizes LPS in whole blood in the absence of anticoagulants. Further studies of its protective efficacy in animal models are warranted. Caution should be used in interpreting assays that measure the binding and neutralization of LPS in whole blood in the presence of calcium-binding anticoagulants or heparin.

Animals↗

PubMed1996

Role of the V2, V3, and CD4-binding domains of GP120 in curdlan sulfate neutralization sensitivity of HIV-1 during infection of T lymphocytes.

A sulfated polysaccharide, curdlan sulfate (CRDS) with 1,3-beta-D-glucan as a main chain, inhibits HIV-1 infection of human peripheral blood lymphocytes (PBLs) by binding to the V3 region of gp 120. We previously showed that T cell (T)-tropic HIV-1 isolates are over 10-fold more sensitive to neutralization by CRDS than macrophage (MT)-tropic viruses, which possesses a relatively less charged amino acid composition in the V3 sequence. To analyze the interaction of CRDS with V3 and its association with neutralization sensitivity of HIV-1 isolates, we examined the effect of CRDS on the binding of neutralizing antibodies to monomeric and oligomeric gp 120 mutants of T- and MT-tropic HIV-1 clones in which the V3 loop was either deleted or substituted by V3 of another isolate. Our results showed that the presence and the amino acid composition of the V3 loop appears to determine the extent of interaction of CRDS with the V2 and CD4-binding regions on native gp 120 monomers; however, the positive charge of V3 has less effect on this interaction on oligomeric gp 120. Furthermore, our results established that only the CRDS-induced masking of V3 on oligomeric gp120 appears to be associated with the anti-HIV-1 activity of CRDS in vitro. Our findings underline the usefulness of CRDS for understanding the structural constraints on gp 120 that drive the transition from MT- to T-tropic isolates in vivo and enable the virus to use multiple fusion cofactors.

Amino Acid Sequence↗

PubMed1995

Design and evaluation of nonpeptide fibrinogen gamma-chain based GPIIb/IIIa antagonists.

Two series of nonpeptide turn mimetics were designed by analysis of the solution NMR structure of the 385-411 sequence of the gamma-chain of fibrinogen. These compounds, based on the KQAGD (Lys-Gln-Ala-Gly-Asp, 406-410) sequence, were synthesized and studied in vitro. The most interesting compound from our study, RWJ 50042 (25), exhibits potent inhibition of fibrinogen binding to GPIIb/IIIa (IC50 = 0.009 microM), as well as thrombin- or collagen-induced platelet aggregation (IC50 = 0.76, 0.14 microM). Since the 400-411 sequence is required for gamma-chain bioactivity and is a unique recognition sequence among ligands for integrins, vis-a-vis other RGD (Arg-Gly-Asp)-presenting proteins, these turn mimetics may represent a new, selective approach to antagonism of the fibrinogen receptor.

Amino Acid Sequence↗

PubMed1994

Mechanism of the inhibitory effect of curdlan sulfate on HIV-1 infection in vitro.

To study the mechanism by which sulfated polysaccharides with 1,3-beta-D-glucan as a main chain exert anti-HIV-1 activity, we analyzed the effects of curdlan sulfate (CRDS) on HIV-1 infection of SupT-1 cells and peripheral blood mononuclear cells. CRDS had no effect on virions inhibited weakly HIV-1 attachment to cells, and had to be present for 24 hr to achieve protection. Lack of HIV-1 DNA corresponding to the gag region in cells incubated with the virus and CRDS and inhibition of infection after addition of 2',3'-dideoxyinosine to cells treated with CRDS and HIV-1 for less than 24 hr suggest that CRDS delays events that precede and/or include reverse transcription. Analysis of the effect of CRDS on binding of HIV-1 neutralizing antibodies to gp 120 demonstrated that both the continuous epitopes on the V3 loop and the discontinuous CD4 binding site of gp 120 represent targets for CDRS. This interaction of CRDS with functional gp 120 domains suggests that CRDS interferes with the membrane fusion process during HIV-1 infection. Concentrations of CRDS that were protective against infection with T cell- and macrophage-tropic HIV-1 isolates had less suppressive effects on T cell function in comparison with the related compound, dextran sulfate.

Anions↗

PubMed1994

Synthetic peptides that mimic the binding site of horseshoe crab antilipopolysaccharide factor.

Tachypleus antilipopolysaccharide (LPS) factor (TALF) is a protein of 102 amino acids in the lysate of amebocytes of Tachypleus tridentatus that binds bacterial LPS with high affinity and blocks its biologic activity in numerous assays. To elucidate the minimal sequences that bind LPS, overlapping synthetic peptides based on the sequence of TALF were assessed for the ability to bind and neutralize LPS. TALF41-53 was the minimal sequence that bound LPS, as assessed by a slot blot capture assay. TALF29-59 bound LPS with the highest potency. TALF29-59 decreased LPS-induced coagulation of limulus amebocyte lysate, induction of cytokines from human monocytes, and LPS-induced lethality in sensitized mice. Synthetic peptides based on TALF or other LPS-binding proteins may be useful for the design of drugs for treatment of endotoxemia.

Amino Acid Sequence↗

PubMed1994

Disintegrins and other naturally occurring antagonists of platelet fibrinogen receptors.

Disintegrins represent a family of low-molecular weight, cysteine-rich, RGD-containing peptides that inhibit fibrinogen binding to glycoprotein IIb/IIIa complex as well as binding of other ligands to RGD-dependent integrins on the surface of other cells. Disintegrins occur in the venom of various vipers. Disintegrin domains have been identified in viper venom hemorrhagins and in sperm proteins involved in the sperm-egg fusion. Amino acid sequences of 25 disintegrins, alignment of S-S bridges in four disintegrins, and stereo models of five disintegrins are presented. Primary structures of disintegrins differ significantly from other fibrinogen receptor antagonists occurring in the venoms of Elapidae, leeches, and ticks. Several aspects of structure-function relationship of disintegrins, their biological activities, and possible clinical applications are discussed.

Amino Acid Sequence↗

PubMed1991

Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces.

Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization (distribution, orientation, conformation) of fibrinogen adsorbed on a hydrophobic surface, we studied the binding of polyclonal and monoclonal antifibrinogen antibodies to polyalkyl methacrylate polymers previously exposed to purified fibrinogen solution or diluted plasma and compared the results with platelet retention in methacrylate bead columns. There was an increase in platelet retention following diluted plasma pretreatment, which was eliminated by a polyclonal antibody against fibrinogen or against a gamma-(395-411) peptide from fibrinogen and was reduced by monoclonal antibodies (4A5, 4-2) against other COOH-terminal gamma-chain epitopes. Monoclonal antibody 10E5 against the fibrinogen receptor GpIIb/IIIa totally inhibited platelet retention in the bead columns. Our data suggest that different methacrylate polymers induce different changes in adsorbed fibrinogen, which may interfere with its interaction with platelets, and that platelet retention in a methacrylate bead column involves interaction of the COOH-terminal end of the gamma-chain of adsorbed fibrinogen with platelet GpIIb/IIIa receptors.

Adsorption↗

PubMed1990

1H NMR sequential assignments and secondary structure analysis of human fibrinogen gamma-chain C-terminal residues 385-411.

The human fibrinogen gamma-chain, C-terminal fragment, residues 385-411, i.e., KIIPFNRLTIGEGQQHHLGGAKQAGDV, contains two biologically important functional domains: (1) fibrinogen gamma-chain polymerization center and (2) platelet receptor recognition domain. This peptide was isolated from cyanogen bromide degraded human fibrinogen and was investigated by 1H NMR (500 MHz) spectroscopy. Sequence-specific assignments of NMR resonances were obtained for backbone and side-chain protons via analysis of 2D NMR COSY, double quantum filtered COSY, HOHAHA, and NOESY spectra. The N-terminal segment from residues 385-403 seems to adopt a relatively fixed solution conformation. Strong sequential alpha CH-NH NOESY connectivities and a continuous run of NH-NH NOESY connectivities and several long-lived backbone NH protons strongly suggest the presence of multiple-turn or helix-like structure for residues 390 to about 402. The conformation of residues 403-411 seems to be much less constrained as evidenced by the presence of weaker and sequential alpha CH-NH NOEs, the absence of sequential NH-NH NOEs, and the lack of longer lived amides. Chemical shifts of resonances from backbone and side-chain protons of the C-terminal dodecapeptide, residues 400-411, differ significantly from those of the parent chain, suggesting that some preferred C-terminal conformation does exist.

Amino Acid Sequence↗

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↗

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

Expression of primary polymerization sites in the D domain of human fibrinogen depends on intact conformation.

Fragments D1 and DD, plasmic degradation products of human fibrinogen and cross-linked fibrin, respectively, originate from the COOH-terminal domain of the parent molecule. Since a specific binding site for fibrin resides in the COOH-terminal region of the gamma chain, the primary structure of the two fragments was compared and their affinity for fibrin monomer measured. Fragments D1 and DD contained the same segments of the three fibrinogen chains, corresponding to the sequences alpha 105-206, beta 134-461, and gamma 63-411. Fragment DD had a double set of the same chain remnants. Fragments D1 and DD inhibited polymerization of fibrin monomer in a dose-dependent manner; 50% inhibition occurred at a molar ratio of fragment to monomer of 1:1 and 0.5:1, respectively. To prevent fibrin monomer polymerization and render it suitable for binding studies in the liquid phase, fibrinogen was decorated with Fab fragments isolated from rabbit antibodies to human fragment D1. Fibrinogen molecules decorated with 6 molecules of this Fab fragment did not clot after incubation with thrombin, and the decorated fibrin monomer could be used to measure binding of fragments D1 and DD in a homogeneous liquid phase. The data analyzed according to the Scatchard equation and a double-reciprocal plot gave a dissociation constant of 12 nM for fragment D1 and 38 nM for fragment DD. There were two binding sites/fibrin monomer molecule for each fragment. After denaturation in 5 M guanidine HCl, the inhibitory function on fibrin polymerization was irreversibly destroyed. Denatured fragments also lost binding affinity for immobilized fibrin monomer. The preservation of the native tertiary structure in both fragments was essential for the expression of polymerization sites in the structural D domain.

Amino Acid Sequence↗

PubMed1986

Platelet aggregation by fibrinogen polymers crosslinked across the E domain.

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg-Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg-Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti-IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.

Adenosine Diphosphate↗

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

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

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.

Blood Platelets↗

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↗