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Biomedical subjects

J Hawiger

Publications and source records attributed to J Hawiger.

At least 55 records · Page 3Linked to original sources
PubMed1989

Fibrinogen-mediated epidermal cell migration: structural correlates for fibrinogen function.

Previously we showed that epidermal cells are able to use fibrinogen (FGN) as a migration substratum during wound closure. The goal of the present study was to determine the structural features of FGN that allow this migration. Pieces of glass coated with native, fragmented, or other modified forms of FGN were implanted into full-thickness skin wounds of adult newts such that migrating epidermal cells would encounter the implant. In this system, a coating of FGN allowed considerably more migration than a coating of BSA. At high concentrations, heat-denatured FGN supported as much migration as the same amount of intact FGN. Fraction I-9, a circulating form of FGN missing a 20-30K (K = 10(3) Mr) carboxy-terminal segment of the A alpha chain, was no less effective than intact FGN. Comparison of the isolated D1 and E fragments of FGN showed migration only on D1, but never to the extent seen on intact FGN containing the same amount of D1. Plasmin digestion of D1 in the presence of EDTA, a process which produces D3, a fragment differing from D1 by the loss of the carboxy-terminal 109 amino acids of the gamma chain, caused a significant loss of activity in the D fragment. Migration was good on implants coated with relatively high concentrations of purified A alpha chains but gamma chains were inactive. Migration over intact FGN was almost totally blocked by 230 microM-Arg-Gly-Asp-Ser (RGDS), a peptide known to interact with integrin-type receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
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↗
PubMed1987

Formation and regulation of platelet and fibrin hemostatic plug.

Formation of a hemostatic plug represents one of the earliest responses to vessel wall injury. Platelets react to any discontinuity in the vascular endothelium through initial contact, spreading, and formation of a thrombus (or aggregate). This development of a primary hemostatic plug requires platelet membrane receptors through which the adhesive macromolecules, von Willebrand factor (vWF) and fibrinogen, anchor platelets to the vessel wall and link them to each other. There are two receptor pathways--classic and alternative--for the binding of vWF to platelets; the latter induced by thrombin, and adenosine diphosphate (ADP) is shared with fibrinogen. Synthetic peptides, patterned after known binding domains of adhesive molecules, have been designed to inhibit their interactions with platelet receptors. A secondary hemostatic plug, composed of platelets enmeshed in fibrin, results from the action of thrombin, which is not only essential for formation of fibrin but also for exposure of platelet receptors for adhesive molecules and for "activation" of factors V and VIII. Thrombin generation is greatly enhanced through the activity of the prothrombinase complex formed on the surface of platelets, perturbed endothelial cells, and leukocytes. A pivotal event is activation of factor X through the intrinsic and extrinsic coagulation pathways. Binding of factors IXa and VIIa to the vascular endothelium represents a localized mechanism for factor Xa generation. Formation of a platelet and fibrin thrombus is controlled by regulatory mechanism: prostacyclin, endogenous heparin-antithrombin III complex, thrombomodulin-protein C-protein S system, and the fibrinolytic system. The balance of all components--vessel wall, platelets, adhesive and coagulation proteins, regulatory mechanisms--determines the effectiveness of the hemostatic plug in maintaining the structural and functional integrity of the circulatory system. An approach to detection of hemostatic derangements in patients at risk evolves from a full understanding of inherited and acquired deficiencies affecting each step of hemostatic plug formation and from selective use of laboratory tests.

Blood Coagulation Tests↗
PubMed1987

Macromolecules that link platelets following vessel wall injury.

Adhesive molecules are essential for anchoring platelets to the zone of vascular injury and for linking them together. Among adhesive molecules, von Willebrand factor and fibrinogen bind to platelets "on demand" when their membrane receptors, composed of membrane glycoproteins, are transformed into the binding mode. At least one receptor mechanism for fibrinogen and for vWF is controlled by ADP that is secreted through the known pathways of platelet activation and counterbalanced by cyclic AMP. Structural and functional studies of adhesive macromolecules led to delineation of receptor pathways responsible for the interaction of platelets with the injured vessel wall and with each other. Synthetic peptide analogues of platelet receptor recognition domains evolved from these studies as a new class of inhibitors of platelet aggregation.

Adenosine Diphosphate↗
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↗
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↗
PubMed1983

Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man.

To assess the pharmacologic effects of aspirin on endogenous prostacyclin and thromboxane biosynthesis, 2,3-dinor-6-keto PGF1 alpha (PGI-M) and 2,3-dinor-thromboxane B2 (Tx-M) were measured in urine by mass spectrometry during continuing administration of aspirin. To define the relationship of aspirin intake to endogenous prostacyclin biosynthesis, sequential urines were initially collected in individuals prior to, during, and subsequent to administration of aspirin. Despite inter- and intra-individual variations, PGI-M excretion was significantly reduced by aspirin. However, full mass spectral identification confirmed continuing prostacyclin biosynthesis during aspirin therapy. Recovery of prostacyclin biosynthesis was incomplete 5 d after drug administration was discontinued. To relate aspirin intake to indices of thromboxane biosynthesis and platelet function, volunteers received 20 mg aspirin daily followed by 2,600 mg aspirin daily, each dose for 7 d in sequential weeks. Increasing aspirin dosage inhibited Tx-M excretion from 70 to 98% of pretreatment control values; platelet TxB2 formation from 4.9 to 0.5% and further inhibited platelet function. An extended study was performed to relate aspirin intake to both thromboxane and prostacyclin generation over a wide range of doses. Aspirin, in the range of 20 to 325 mg/d, resulted in a dose-dependent decline in both Tx-M and PGI-M excretion. At doses of 325-2,600 mg/d Tx-M excretion ranged from 5 to 3% of control values while PGI-M remained at 37-23% of control. 3 d after the last dose of aspirin (2,600 mg/d) mean Tx-M excretion had returned to 85% of control, whereas mean PGI-M remained at 40% of predosing values. Although the platelet aggregation response (Tmax) to ADP ex vivo was inhibited during administration of the lower doses of aspirin the aggregation response returned to control values during the final two weeks of aspirin administration (1,300 and 2,600 mg aspirin/d) despite continued inhibition of thromboxane biosynthesis. These results suggest that although chronic administration of aspirin results in inhibition of endogenous thromboxane and prostacyclin biosynthesis over a wide dose range, inhibition of thromboxane biosynthesis is more selective at 20 than at 2,600 mg aspirin/d. However, despite this, inhibition of platelet function is not maximal at the lower aspirin dosage. Doses of aspirin in excess of 80 mg/d resulted in substantial inhibition of endogenous prostacyclin biosynthesis. Thus, it is unlikely that any dose of aspirin can maximally inhibit thromboxane generation without also reducing endogenous prostacyclin biosynthesis. These results also indicate that recovery of endogenous prostacyclin biosynthesis is delayed following aspirin administration and that the usual effects of aspirin on platelet function ex vivo may be obscured during chronic aspirin administration in man.

Adenosine Diphosphate↗
PubMed1982

Evidence that changes in platelet cyclic AMP levels regulate the fibrinogen receptor on human platelets.

Fibrinogen binds to human platelets after specific receptor sites are exposed by thrombin, ADP, epinephrine, and other stimuli. Since prostaglandin I2 (PGI2), a potent activator of platelet adenylate cyclase, prevents mobilization of the fibrinogen receptor by aggregating agents, we investigated the relationship between platelet cAMP levels and fibrinogen receptor status in thrombin-stimulated human platelets. A dose-dependent rise in platelet cAMP in response to two adenylate cyclase agonists, PGI2 and forskolin, correlated with progressive inhibition of fibrinogen binding. Moreover, the receptor inhibition produced by either agonist was sustained up to 2 h and was associated with a persistent increase in cAMP levels. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, in the presence of a subthreshold concentration of PGI2 also raised cAMP and inhibited fibrinogen binding. In contrast, the effects of PGI2 on both cAMP and fibrinogen binding were markedly attenuated by 9-(tetrahydro-2-furyl) adenine, an adenylate cyclase inhibitor. These results indicate that the inhibition of fibrinogen binding by PgI2 is linked to its effect on cAMP levels and suggest that elevation of platelet cAMP levels from any cause prevents exposure of the fibrinogen receptor.

Blood Platelets↗