Differential binding of mannose-specific lectins to the carbohydrate chains of fibrinogen domains D and E

The interaction of fibrinogen with the mannose-specific lectins concanavalin A (ConA), its acetyl derivative (Ac-ConA) and Lens culinaris agglutinin (LcH) was studied. Both ConA and LcH interact specifically with individual fibrinogen B beta and gamma chains and with denatured fragments D and E. However, analysis of the binding data shows that four moles of Ac-ConA are bound per mole of fibrinogen with two sets of binding sites (Kd1 = 2.4 microM and Kd2 = 16.6 microM; n1 = n2 = 2) while only two moles of LcH are bound per mole of fibrinogen (Kd = 2.6 microM). Ultracentrifugation studies are also in agreement with the presence in the fibrinogen molecule of two and four binding sites for LcH and Ac-ConA, respectively. No aggregates of fibrinogen formed through LcH or Ac-ConA linkages are observed. The use of a crosslinking reagent and ultracentrifugal analysis of the lectin-fibrinogen fragments D1 and E complexes indicated that ConA, as well as Ac-ConA, interact with both fragments D and E while LcH interacts only with fragment D. Furthermore, the binding of ConA to both D and E domains in the intact fibrinogen molecule is clearly demonstrated by using a bifunctional reagent. The bivalent character of ConA tetramers may be misinterpreted as a lack of accessibility of the lectin to two of the four carbohydrate chains of fibrinogen. The differential binding of LcH and ConA to the carbohydrate chains of fibrinogen can be related to a different exposure of the oligosaccharide in D and E fragments and domains and to the different requirements of both lectins for their binding to glycoproteins.     

Plasminogen activation in diabetes mellitus. Kinetic analysis of plasmin formation using components isolated from the plasma of diabetic donors

Two components of the fibrinolytic system, plasminogen and the vascular plasminogen activator, have been isolated to apparent homogeneity from the post-venous occlusion plasma of three diabetic patients (hemoglobin A1C greater than 7%) and of one nondiabetic control person. Plasminogen activation was studied for each person separately in the absence and presence of CNBr fragments of fibrinogen. Activation of diabetic plasminogen by urokinase was not significantly altered as compared to the activation of control plasminogen. The same was found when diabetic plasminogen was activated by control vascular plasminogen activator in the presence of fibrinogen fragments but only at plasminogen concentrations below 10-30 nM; at higher substrate concentrations, however, plasminogen activation was impaired in a pattern resembling substrate inhibition. Activation of control plasminogen by diabetic vascular plasminogen activator was completely impaired in the absence of fibrinogen fragments. Addition of fibrinogen fragments stimulated plasmin formation by diabetic vascular plasminogen activator resulting in kinetic constants which were similar to the activation of control plasminogen by control vascular plasminogen activator in the absence of fibrinogen fragments (Km = 7.5 microM, kcat = 0.05 S-1). Addition of fibrinogen fragments in controls decreased Km values to less than 0.1 microM. Despite addition of fibrinogen fragments the rate of plasmin formation from diabetic plasminogen by diabetic vascular plasminogen activator isolated from the same diabetic donor was so small that kinetic constants could not be calculated.     

Binding and processing of fibrinogen by rabbit hepatocytes

We describe a specific fibrinogen-hepatocyte interaction. Rabbit 125I-labeled fibrinogen (125I-FGN) was incubated at 4 degrees C with suspensions of rabbit hepatocytes (approximately 1 X 10(6) cells/ml). Bound ligand was separated from free by centrifugation of cells through oil and quantitated by gamma-scintillation counting. Specific binding, determined by subtraction of nonspecific binding in the presence of 8 mM EDTA from total binding in the presence of 2 mM CaCl2, required 3 h to plateau and represented approximately 70% of total binding. Specific binding was calcium-dependent and was negligible in buffer containing 2 mM MgCl2. Half-maximal saturation occurred at approximately 30 nM 125I-FGN with approximately 480,000 molecules/cell at saturation. Dilution experiments revealed comparable affinities for labeled and unlabeled fibrinogen. Total binding was irreversible as determined by addition of excess unlabeled fibrinogen or EDTA. Specific binding of 25 nM 125I-FGN was inhibited, in a concentration-dependent fashion, by unlabeled fibrinogen or fibrinogen fragment D95 (Mr = 95,000), but not by fibrinogen fragment E or Arg-Gly-Asp-containing peptides. Unlabeled fibrinogen (3.1 microM) completely abolished specific binding, whereas greater than 80% inhibition was achieved with 10 microM fragment D95. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of 125I-FGN bound in the presence of calcium demonstrated disappearance of A alpha chains with formation of products of Mr greater than 200,000; EDTA or unlabeled fibrinogen prevented fibrinogen processing. These data describe a unique fibrinogen-hepatocyte interaction which differs considerably from the platelet-fibrinogen interaction, especially with regard to the processing of the fibrinogen molecule.     

A turbidimetric assay for quantitating functional fibrin(ogen) using polystyrene-divinylbenzene microparticles.

A sensitive assay has been developed to quantitate fibrinogen in plasma or in other aqueous solutions. Microscopic latex particles, modified with a mixed monomolecular film of lecithin and fibrinogen, are used as a solid-phase reagent. These lecithin/fibrinogen-coated beads aggregate when stirred in the presence of thrombin and, when solution-phase fibrinogen is added, the increased rate of aggregation is proportional to the concentration of soluble fibrinogen. Using a sample volume of 200 microl, as little as 15 nM ( approximately 5 microg ml-1) fibrinogen can be measured. Fibrinogen determinations using the bead assay compared favorably with those derived from a standard clinical assay, with a correlation coefficient (r2) of 0.9710 over a range of 2.5 to 28.0 microM. Analytic precision was comparable to available assays, with typical coefficients of variation of 12.7 and 7.1% for fibrinogen concentrations of 30 nM and 15.0 microM, respectively. The method has a dynamic range of 15 nM to over 3.0 microM and offers the advantage of being sensitive to 20-fold lower concentrations of fibrinogen compared to routine clot-based methods. Unlike immunological assays, e.g., ELISA, it measures only the functional protein. This bead method should prove to be of greatest use to investigators measuring low levels of functional fibrin(ogen).     

Induction of the fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP

The capacity of epinephrine alone and the combination of low dose epinephrine and ADP to support the binding of fibrinogen to washed human platelets has been examined, 125I-Fibrinogen was bound to epinephrine-stimulated platelets, but 90 min were required to achieve maximal binding at 22 degrees C in contrast to 20 to 30 min with ADP. The overall rate of interaction appeared to reflect the slow binding of fibrinogen to epinephrine-stimulated platelets as opposed to the rate of stimulation of the cell. Divalent ions were required for binding of fibrinogen to epinephrine-stimulated platelets, and both calcium and magnesium supported binding with a prolonged time course. Fibrinogen binding was maximally supported by 20 to 30 microM epinephrine. The combination of low dose epinephrine (5 microM) and low dose ADP (0.5 microM), which acted synergistically to induce platelet aggregation, supported the rapid (10 min) binding of fibrinogen to platelets. With 4 microM epinephrine, more fibrinogen bound per platelet at all ADP doses than with ADP alone. With all the stimuli, saturable binding of fibrinogen to the platelet was observed, and Scatchard plots were linear, yielding very similar apparent association constants. The number of molecules bound per cell was stimulus-dependent, with 30 microM epinephrine inducing the binding of fewer fibrinogen molecules per cell (mean = 20,400) than 10 microM ADP (mean = 35,900) or the combination of 5 microM epinephrine + 0.5 microM ADP (mean = 43,600). The participation of endogenous ADP in fibrinogen binding to epinephrine-stimulated platelets was suggested since enzymes which remove ADP, apyrase, and creatine phosphate/creatine phosphokinase, and the ADP analogue, 2-chloroadenosine, completely inhibited the binding of fibrinogen to the platelet.     

Histaminylation of fibrinogen by tissue transglutaminase-2 (TGM-2): potential role in modulating inflammation

Plasma fibrinogen plays an important role in hemostasis and inflammation. Fibrinogen is converted to fibrin to impede blood loss and serves as the provisional matrix that aids wound healing. Fibrinogen also binds to cytokine activated endothelial cells and promotes the binding and migration of leukocytes into tissues during inflammation. Tissue transglutaminase (TGM-2) released from injured cells could cross-link fibrinogen to form multivalent complexes that could promote adhesion of platelets and vascular cells to endothelium. Histamine released by mast cells is a potent biogenic amine that promotes inflammation. The covalent attachment of histamine to proteins (histaminylation) by TGM-2 could modify local inflammatory reactions. We investigated TGM-2 crosslinking of several biogenic amines (serotonin, histamine, dopamine and noradrenaline) to fibrinogen. We identified histaminylation of fibrinogen by TGM-2 as a preferred reaction in solid and solution phase transglutaminase assays. Histamine caused a concentration-dependent inhibition of fibrinogen cross-linking by TGM-2. Fibrinogen that was not TGM-2 crosslinked bound to un-activated endothelial cells with low affinity. However, the binding was increased by sevenfold when fibrinogen was cross-linked by TGM-2. Histaminylation of fibrinogen also inhibited TGM-2 crosslinking of fibrinogen and the binding to un-activated HUVEC cells by 75–90 %. In summary, the histaminylation of fibrinogen by TGM-2 could play a role in modifying inflammation by sequestering free histamine and by inhibiting TGM-2 crosslinking of fibrinogen.     

The effect of the single substitution of arginine within the RGD tripeptide motif of a modified neurotoxin dendroaspin on its activity of platelet aggregation and cell adhesion

The Arg-Gly-Asp (RGD) tripeptide unit is a cell-cell and cell-extracellular matrix recognition sequence of some integrins that is found within several extracellular matrix glycoproteins and dendroaspin, a disintegrin-like venom protein isolated from the snake venom of the Dendroaspis jamsonii. In the present study, the RGD motif in dendroaspin was substituted by Lys-Gly-Asp (KGD), His-Gly-Asp (HGD), Gln-Gly-Asp (QGD) and Ala-Gly-Asp (AGD) denoted as KGD-den, HGD-den, QGD-den and AGD-den, respectively. Each of the mutants exhibited activity as inhibitor of ADP-induced platelet aggregation with IC50 values of 0.26, 2.5, 6, and 17 microM for KGD-den, HGD-den, QGD-den, and AGD-den, respectively, as compared with RGD-den (IC50 = 0.18 microM). Interestingly, HGD-den was approx. two-fold more potent and a more selective inhibitor than either the KGD-den or QGD-den counterpart at blocking A375-SM human melanoma cell adhesion to fibrinogen (beta3-mediated). KGD-den, HGD-den, and QGD-den were preferentially antagonists of A375-SM human melanoma cell adhesion to fibrinogen rather than to fibronectin (alpha5beta1-, beta3-mediated). Both HGD-den and KGD-den were equipotent as inhibitors of human erythroleukaemia (HEL) cell adhesion to fibrinogen (IC50 = 0.15 microM) and also preferential inhibitors of HEL cell adhesion to fibrinogen (beta3 and beta1-mediated) rather than to fibronectin. These findings show that the presence of the arginine within the RGD motif of dendroaspin is not obligatory and substitution of this residue can modulate inhibitory potency and integrin binding selectivity.     

Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator

The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin.     

Identification of a monoclonal antibody against platelet GPIIb that interacts with a calcium-binding site and induces aggregation.

We have characterized a monoclonal antibody named D33C, specific for platelet glycoprotein (GP) IIb, which induces fibrinogen binding and platelet aggregation. D33C Fab fragments interact with an average of 44,000 +/- 20,000 sites on resting platelet with a Kd value of 0.8 microM. This value decreased to 0.17 microM in the presence of 1 mM EDTA suggesting that Ca2+ chelation increases the antibody affinity. Purified IgGs and Fab fragments exhibit a similar potency and induce binding of fibrinogen and aggregation at levels comparable to those obtained with ADP. D33C-induced platelet aggregation, however, was not inhibited by 1 microM PGE1 and was not associated with a significant [14C]serotonin release, suggesting differences with ADP in the mechanism of activation. Among a large series of synthetic peptides corresponding to potential antigenic sequences within the structure of GPIIb, one peptide with the sequence DIDDNGYPDLIV was found to inhibit D33C activity. This peptide corresponds to a putative calcium-binding site whose sequence is highly homologous to similar sequences present in the alpha subunits of the fibronectin and the vitronectin receptors. Despite this homology, D33C interacts only with platelet GPIIb suggesting that the identified epitope may be differently exposed at the surface of the cells. This antibody may prove to be a valuable tool to study the induction reaction on recombinant GPIIbIIIa expressed in cells that lack the appropriate signal transduction reactions.     

The effects of X rays on BCNU-induced DNA crosslinking

We have used the technique of alkaline elution to study DNA interstrand crosslinking in 9L rat brain tumor cells treated with combinations of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and X rays. Irradiation with doses as low as 50 rad of X rays immediately or 6 hr after a 1-hr treatment with 60, 80, or 100 microM BCNU produced a significant increase in BCNU-induced DNA interstrand crosslinking. If cells were irradiated before BCNU treatment, the amount of crosslinking was not affected compared with BCNU alone. Cell survival experiments using 600 rad of X rays and 1-hr treatments with 0-30 microM BCNU were also performed. As found in the crosslinking studies, irradiation immediately or 6 hr after the BCNU treatment produced enhanced cell kill, but irradiation 6 hr before BCNU treatment did not produce enhanced cell kill. Therefore, the X-ray-mediated increase in BCNU-induced DNA interstrand crosslinking may be the mechanism through which cell kill is increased by combination treatment with the agents.     

Thrombospondin interaction with fibrinogen. Evidence for binding to the A alpha- and B beta-chains of fibrinogen

Platelet thrombospondin interacts with fibrinogen in a specific and saturable manner. Thrombospondin was found to specifically bind to the A alpha- and B beta-chains of fibrinogen; binding was independent of divalent ions. Binding could be blocked either by preincubation of thrombospondin with 9.4 microM fibrinogen or by preincubation of fibrinogen with 1.1 nM thrombospondin. Thrombospondin bound only to the beta-chain component of the D and DD plasmin fragment of fibrinogen. Thrombospondin interaction with fibrinogen was not blocked by preincubation with synthetic peptides which have previously been identified as either the fibrinogen receptor (alpha 572-575, the synthetic tetrapeptide arginyl-glycyl-aspartyl-serine) or cell attachment (gamma 400-411) domains. Fibrinogen, therefore, possesses at least two unique and distinct sites, within the A alpha- and B beta-chains, for its interaction with thrombospondin.     

Soluble fibrin consists of fibrin oligomers of heterogeneous distribution

Soluble fibrin is observed in patients with intravascular coagulation and represents an intermediary product of conversion of fibrin monomers into a fibrin clot whereby the presence of fibrinogen may suppress fibrin clot formation. The interactions between fibrin and fibrinogen and the occurrence of fibrin oligomers in soluble fibrin were studied by sucrose density ultracentrifugation. Different concentrations of soluble fibrin, prepared by mixing 125I-fibrin (24 nM - 1.5 microM) with a constant concentration of 131I-fibrinogen (6 microM) were analyzed at 37 degrees C in stable linear sucrose density gradients containing a uniform concentration of unlabelled fibrinogen (6 microM) and calcium ions in order to mimic the physiological situation. At any fibrin concentration, 125I-fibrin sedimented faster than 131I-fibrinogen through 5-30% (w/v) sucrose gradients. Sedimentation rates of fibrin increased from 9 S to 23 S depending on the initial fibrin concentration. The relative amount of residual fibrin monomer not incorporated into oligomers was calculated from the sedimentation profiles. At any fibrin concentration, the portion of free monomer was always more than twofold higher for batroxobin-generated (desAA-) fibrin than for thrombin-generated (desAABB-) fibrin. Apparent association constants for desAABB-fibrin were 3-10 times higher than those for desAA-fibrin indicating a stronger interaction between monomers of the former type of fibrin. In the presence of excess fibrinogen the predominant species in soluble desAA-fibrin were monomers and dimers, whereas dimers, trimers and higher-molecular-mass oligomers were present in soluble desAABB-fibrin. Strong interactions between both types of fibrin were demonstrated from their cosedimentation, whereby the size of these copolymers were shown to be governed by the oligomer size of the desAABB-fibrin type. These results provide evidence for the occurrence of differently sized oligomers of fibrin in soluble fibrin and for the concept of a cooperative polymerization process between both types of fibrin devoid of any stable complexes between fibrin and fibrinogen.     

Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex

The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.     

Regulation of the streptokinase-mediated activation of human plasminogen by fibrinogen and chloride ions

The effects of human fibrinogen and Cl- on the activation of native human plasminogen by streptokinase, at 37 degrees C, in isotonic buffers, consisting of 10 mM Hepes-NaOH, 150 mM NaOAc-NaCl, pH 7.4, have been examined. We find that Cl- acts as a mixed-type inhibitor of plasminogen activation, with a Ki in the range of 6.4-9.2 mM, in the absence and presence (1.0 microM) of fibrinogen. Fibrinogen displays an effect on plasminogen activation kinetics consistent with its function as a mixed-type nonessential activator, with Ka values between 110 and 240 nM, in the absence and presence (50 mM) of Cl-. These observations suggest opposing roles for fibrinogen and Cl- in regulating plasminogen activation.     

Factor XIII-induced crosslinking in solutions of fibrinogen and fibronectin

In solutions containing fibrinogen and fibronectin, factor XIIIa catalyzes the formation of two types of crosslinked polymers: hybrid oligomers consisting of equimolar amounts of fibrinogen and fibronectin, and fibrinogen oligomers. The two types of oligomers are produced in amounts proportional to the starting concentration of fibronectin and fibrinogen in the reaction mixture. Increasing the fibronectin concentration relative to the fibrinogen concentration results in the production of more hybrid and less fibrinogen type oligomers. The lowest molecular weight hybrid oligomer, a dimer, is formed by ligation of one molecule of fibrinogen and fibronectin. The A alpha-chain of fibrinogen and one fibronectin subunit participate in the crosslinking. Larger size hybrid oligomers form by the joining of two hybrid dimers to each other via gamma-chain dimerization in the fibronectin moiety of the dimers. In fibrinogen oligomer formation, fibrinogen molecules are ligated by gamma-chain dimerization in a step-wise fashion producing fibrinogen dimers, trimers, tetramers, etc. without A alpha-chain crosslinking. The hybrid type and the fibrinogen type of oligomer grow in size and eventually become crosslinked to each other yielding large molecular weight complexes that interact to form a gel network.     

A thrombin-based peptide corresponding to the sequence of the thrombomodulin-binding site blocks the procoagulant activities of thrombin.

Thrombomodulin, a cofactor in the thrombin-catalyzed activation of protein C, blocks the procoagulant activities of thrombin such as fibrinogen clotting, Factor V activation, and platelet activation. The binding site for thrombomodulin within human thrombin has been localized at a region comprising residues Thr147-Ser158 of the B-chain of thrombin. The dodecapeptide sequence, TWTANVGKGQPS, corresponding to these residues inhibits thrombin binding to thrombomodulin with an apparent Ki = 94 microM (Suzuki, K., Nishioka, J., and Hayashi, T. (1990) J. Biol. Chem. 265, 13263-13267). We have found that the inhibitory effect of the dodecapeptide on the thrombin-thrombomodulin interaction is sequence-specific, and that residues Asn151, Lys154, and Gln156 are essential for thrombomodulin binding. The dodecapeptide was also found to directly block thrombin procoagulant activities, fibrinogen clotting (concentration for half-maximum inhibition, 385 microM). Factor V activation (concentration for half-maximum inhibition, 33 microM), and platelet activation (concentration for half-maximum inhibition, 645 microM). This peptide did not block thrombin inhibition by antithrombin III, but blocked thrombin inhibition by hirudin. These findings suggest that the binding site for thrombomodulin in thrombin is shared with the sites for fibrinogen, Factor V, platelets, and hirudin, and that, therefore, the inhibition of thrombin procoagulant activities by thrombomodulin in part results from blocking of the interaction between thrombin and the procoagulant protein substrates by thrombomodulin.     

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.     

Characteristics of collagen-induced fibrinogen binding to human platelets

Polymerized type I calf skin collagen induced a time-dependent specific binding of 125I-fibrinogen to washed human platelets. Binding occurred more rapidly in a shaken rather than in an unstirred system. It was linear in the range 0.05-0.3 microM added fibrinogen and was saturated at higher fibrinogen concentrations (more than 0.8 microM). Scatchard analysis showed a single population of binding sites (16530 +/- 5410 per platelet) with a Kd = 0.53 +/- 0.23 microM. Collagen-induced 125I-fibrinogen binding to platelets was completely inhibited by ADP antagonists such as creatine phosphate/creatine phosphokinase and AMP, and partially inhibited by pretreatment of the platelets with aspirin. With both normal and aspirin-treated platelets a close correlation was observed between the amount of 125I-fibrinogen bound and the extent of dense granule secretion. Our results confirm that fibrinogen becomes bound to platelet surface receptors during collagen-induced platelet aggregation and suggest that secreted ADP is an essential cofactor in this process.     

Effects of human fibrinogen and its cleavage products on activation of human plasminogen by streptokinase

The influence of human fibrinogen (Fg) and its terminal plasminolytic digestion products, fragment D and fragment E, on the kinetics of activation of human plasminogen (Pg) by catalytic levels of streptokinase (SK) has been investigated. Both Fg and fragment D enhanced the rates of activation of human Glu1-Pg, Lys77-Pg, and Val442-Pg. Fragment E was refractive in this regard. In the case of Glu1-Pg, the Km for activation by SK, 0.4 microM, was not affected by the presence of Fg or fragment D. The kcat for this same reaction, 0.12 s-1, was elevated to 0.3 s-1 at saturating levels of these effector molecules. On the other hand, the Km for activation of Lys77-Pg, 0.5 microM, was decreased to 0.09 microM, whereas the kcat, 0.33 s-1, was not altered in the presence of saturating concentrations of Fg or fragment D. In the case of Val442-Pg, the Km for this same activation, 2.0 microM, was lowered to 0.4 microM and 0.25 microM in the presence of Fg and fragment D, respectively. The kcat for this process, 1.0 s-1, was unchanged in the presence of these agents. The concentrations of Fg (KFg) and fragment D (KFD) that led to half-maximal stimulation of the activation rates were determined. For Fg with Glu1-Pg, Lys77-Pg, and Val442-Pg, the KFg values were 0.08 microM, 0.14 microM, and 0.17 microM, respectively. The KFD values for these same plasminogens were 0.25 microM, 2.0 microM, and 1.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)