Exposure of platelet fibrinogen receptors by a monoclonal antibody to CD9 antigen

We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot-reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti-glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non-stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.     

Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta-thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.     

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.     

Induction of platelet Ca2+ influx and mobilization by a monoclonal antibody to CD9 antigen

We found that a monoclonal antibody (MoAb) to CD9 antigen, PMA2, induced a rise in cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded platelets, and we examined whether this response was due to direct action of PMA2 on CD9 antigen. The rise in [Ca2+]i was dependent on the PMA2 concentration, irrespective of the presence or absence of extracellular Ca2+. The role of secreted adenosine diphosphate (ADP) and thromboxane in the [Ca2+]i response to PMA2 was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. Combined treatment with CP/CPK and aspirin abolished the rise in [Ca2+]i, although either CP/CPK or aspirin alone produced only partial inhibition. Inhibition of adenosine triphosphate (ATP) secretion and thromboxane B2 synthesis by an MoAb to the glycoprotein IIb-IIIa complex, PMA1, resulted in little [Ca2+]i response to PMA2. In contrast, thrombasthenic platelets, in which ATP secretion and thromboxane B2 synthesis were normal, showed a normal [Ca2+]i response. When PMA2 was added to CD9+ mononuclear cells, no rise in [Ca2+]i was observed. Thus, we conclude that binding of monoclonal immunoglobulin G molecules to the CD9 antigen raises [Ca2+]i through the effect of secreted ADP and thromboxane on platelets, and that CD9 antigen is not directly involved in induction of Ca2+ influx and mobilization.     

The effect of thrombin on the dynamic exchange between intraplatelet and extraplatelet fibrinogen

We examined the distribution of platelet fibrinogen and the exchange between intra- and extra-platelet fibrinogen in unstimulated and thrombin-stimulated platelets. In unstimulated platelets 60% of platelet fibrinogen was found in the soluble platelet fraction and 40% in the insoluble one. In platelets activated with thrombin, changes took place in the distribution of intraplatelet fibrinogen but not in the total fibrinogen content. At 0.5 U/ml of thrombin the fibrin(ogen) content of the insoluble and soluble fractions was approximately 80% and 20%, respectively. When we evaluated how extraplatelet fibrinogen affects the content and distribution of intraplatelet fibrinogen, we found that when unlabelled fibrinogen was added to unstimulated and thrombin-stimulated platelets the content and distribution of intraplatelet fibrinogen remained unaltered. However, when ¹²⁵I-fibrinogen was added, it was incorporated into unstimulated and thrombin-stimulated platelets. In unstimulated platelets, 70% of the incorporated ¹²⁵I-fibrinogen was in the soluble fraction and 30% in the insoluble. In thrombin-stimulated platelets the distribution of the incorporated ¹²⁵I-fibrinogen was 62% and 38% in soluble and insoluble fractions respectively. MoAb to GPIIb–IIIa produced 80% and 60% inhibition of ¹²⁵I-fibrinogen incorporation by unstimulated and thrombin-stimulated platelets. Our data showed dynamic exchange between intraplatelet and extraplatelet fibrinogen both in unstimulated and thrombin-stimulated platelets mediated mainly by GPIIb–IIIa.     

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.     

The effect of thrombin on the dynamic exchange between intraplatelet and extraplatelet fibrinogen

We examined the distribution of platelet fibrinogen and the exchange between intra- and extra-platelet fibrinogen in unstimulated and thrombin-stimulated platelets. In unstimulated platelets 60% of platelet fibrinogen was found in the soluble platelet fraction and 40% in the insoluble one. In platelets activated with thrombin, changes took place in the distribution of intraplatelet fibrinogen but not in the total fibrinogen content. At 0.5 U/ml of thrombin the fibrin(ogen) content of the insoluble and soluble fractions was approximately 80% and 20%, respectively. When we evaluated how extraplatelet fibrinogen affects the content and distribution of intraplatelet fibrinogen, we found that when unlabelled fibrinogen was added to unstimulated and thrombin-stimulated platelets the content and distribution of intraplatelet fibrinogen remained unaltered. However, when ¹²⁵I-fibrinogen was added, it was incorporated into unstimulated and thrombin-stimulated platelets. In unstimulated platelets, 70% of the incorporated ¹²⁵I-fibrinogen was in the soluble fraction and 30% in the insoluble. In thrombin-stimulated platelets the distribution of the incorporated ¹²⁵I-fibrinogen was 62% and 38% in soluble and insoluble fractions respectively. MoAb to GPIIb–IIIa produced 80% and 60% inhibition of ¹²⁵I-fibrinogen incorporation by unstimulated and thrombin-stimulated platelets. Our data showed dynamic exchange between intraplatelet and extraplatelet fibrinogen both in unstimulated and thrombin-stimulated platelets mediated mainly by GPIIb–IIIa.     

The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets.

The Arg-Gly-Asp sequence resides in the cell attachment region of fibronectin. Arg-Gly-Asp-containing peptides support fibroblast attachment, inhibit fibroblast adhesion to fibronectin, and inhibit fibronectin binding to thrombin-stimulated platelets. In view of the similarities between the binding of fibronectin, fibrinogen, and von Willebrand factor to stimulated platelets, we have examined the effects of Arg-Gly-Asp-containing peptides on the interaction of these latter two adhesive proteins with platelets. Gly-Arg-Gly-Asp-Ser-Pro was used as a prototype peptide, and this hexapeptide inhibited fibrinogen binding to ADP and thrombin-stimulated platelets in the 10-200 microM range. The inhibition exceeded 90% at high concentrations of peptide and was observed in the presence of either calcium or magnesium. Platelet aggregation was also inhibited by the peptide in this dose range. The hexapeptide inhibited fibrinogen binding to platelets with receptors fixed in an exposed state, indicating direct interference with the ligand-platelet interaction. The peptide was 1/2 to 1/3rd as potent in inhibiting fibrinogen as fibronectin binding to platelets, but fibrinogen and von Willebrand factor binding were inhibited to an identical extent. Conservative amino acid substitutions for the arginine, glycine, or aspartic acid markedly reduced inhibitory activity and the Asp-Gly-Arg sequence was inactive. These results indicate that Arg-Gly-Asp-containing peptides can inhibit the binding of the three adhesive proteins to stimulated platelets, establishing a basic common feature between the interaction of these molecules with platelets.     

Interaction of a Monoclonal Antibody to Glycoprotein IV (CD36) with Human Platelets and its Effect on Platelet Function

FA6-152, a monoclonal antibody to platelet membrane glycoprotein IV (CP IV), was used to quantify the expression of this glycoprotein on platelets, as well as to evaluate its role in platelet aggregation. On resting platelets, 19 400 ± 7700 molecules of the (125)I-labelled IgC could bind per platelet (n = 20). Binding was not modified following stimulation of the platelets with ADP (10 μmol/l) or thrombin (0.1 U/ml). Fab fragments prepared from the antibody by papain digestion also bound to the platelet surface in a saturable manner. Both the intact IgC and its Fab fragments were found to inhibit platelet aggregation and secretion induced by ADP or collagen in platelet-rich plasma and by thrombin in platelet suspensions. Under nonstirred conditions, whereby the release reaction was only minimally affected, the antibody markedly inhibited thrombin-induced surface expression of α-granule thrombospondin (TSP), whereas it did not alter the concomitant expression of α-granule fibrinogen. In addition, electron microscopy revealed a predominant distribution of TSP and T;P IV on pseudopodia and between adherent cells on thrombin-stimulated platelets. These findings thus support the hypothesis that the interaction of TSP with GP IV on the platelet surface is required for an optimal platelet aggregation/secretion process to occur.     

Effect of fibrinogen concentration on the velocity of platelet aggregation

The relationship between fibrinogen binding to its receptor and platelet aggregation has been investigated by comparing 125I-fibrinogen binding and aggregation velocities of gel-filtered platelets in the presence of adenosine diphosphate (ADP). Aggregometric responses at various fibrinogen concentrations are found to be bell-shaped and show a maximum at fibrinogen concentrations (Fmax) similar to the 125I-fibrinogen hemisaturating doses. At higher and lower fibrinogen concentrations, platelet aggregation velocities decrease in a parallel manner. Lowering ADP concentration increases Fmax, in agreement with the modulatory effect of ADP on fibrinogen binding to platelets. Variations of fibrinogen in the range of physiopathologic plasma concentrations affect platelet aggregation induced by any ADP dose. These results clarify the relationship between the fibrinogen binding process and aggregation and demonstrate that plasma fibrinogen concentration has a major influence on the velocity of platelet aggregation.     

Nitric Oxide (NO)-Platelet Interactions: Inhibition is Independent of the Prostanoid and ADP Pathways

Effects of nitric oxide (NO) on thrombin-induced responses in gel-filtered, [(32)P] Pi-(pre) labeled platelets (GFP) were examined. NO did not alter the levels of (32)P-labeled polyphosphoinositides in unstimulated platelets and did not inhibit the forskolin-induced elevation of [(32)P]PIP (phosphatidylinositol 4-phosphate), which indicates that NO does not concomitantly increase the level of cAMP in resting human platelets. In aspirinated platelets NO inhibited thrombin (0.05 U/ml)-induced formation of [(32)P]phosphatidic acid (PA), secretion of ATP + ADP from the dense granules and secretion of acid glycosidases in a dose-dependent manner. At 0.2 U/ml of thrombin NO still inhibited these responses, although to a lesser degree. In aspirinated platelets in the presence of creatine phosphate/creatine phosphokinase (CP/CPK) to remove secreted ADP, increasing concentrations of NO still produced strong inhibition of [(32)P] PA-formation and secretory responses.     

Effects of the cell adhesion peptide, Arg-Gly-Asp-Ser, on responses of washed platelets from humans, rabbits, and rats

Fibrinogen is a cofactor in the aggregation of human platelets, and is required for ADP-induced aggregation of washed platelets; however, exogenous fibrinogen is not required for ADP-induced aggregation of washed platelets from rabbits or rats. Because with human platelets the cell adhesion peptide, Arg-Gly-Asp-Ser (RGDS), inhibits aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets, its effects on rabbit and rat platelets were studied to investigate the differences in the fibrinogen requirements of platelets from the three species. RGDS (50 mumol/L) caused greater than 80% inhibition of thrombin- induced or (ADP + fibrinogen)-induced aggregation of human platelets, but only 3% to 9% inhibition of the aggregation of rabbit or rat platelets, regardless of whether fibrinogen was added. RGDS inhibited the binding of 125I-fibrinogen to ADP-stimulated human platelets by 80% to 90%, but by only 15% to 27% in the case of rabbit or rat platelets. The differences were due to the species of platelets, since human and rabbit fibrinogens gave similar results. In addition, RGDS failed to displace fibrinogen from the surface of rabbit platelets that had been stimulated with ADP. Thus, there are species differences in the ability of the cell adhesion peptide, RGDS, to block the platelet fibrinogen receptor, even within the mammalian species.     

An investigation into the role of coagulation factor XIII in ADP- induced aggregation and fibrinogen binding with rabbit platelets

Because there was a possibility that activated factor XIII (factor XIIIa) might stabilize a platelet-fibrinogen aggregate through its crosslinking action, we have isolated plasma factor XIII, activated it, and studied the effect of factor XIIIa at a concentration of 3.3 micrograms/ml on aggregation and 125I-fibrinogen binding of rabbit platelets stimulated with 9 microM ADP. Factor XIIIa did not cause aggregation in the absence of ADP, nor did it enhance ADP-induced aggregation or substantially stabilize the platelet aggregate. The presence of factor XIIIa did not affect the amount of fibrinogen bound to platelets immediately after stimulation with ADP, but it appeared to cause a slow specific binding of 125I-fibrinogen to platelets whether or not they were stimulated with ADP. This binding, which was not inhibited by prostaglandin E1, did not lead to aggregation and was accompanied by crosslinking of fibrinogen through its A alpha and gamma chains, either to other fibrinogen molecules or to a platelet protein or proteins.     

Fibronectin binding to thrombin-stimulated platelets: evidence for fibrin(ogen) independent and dependent pathways

Plasma fibronectin binds in a specific and saturable manner to thrombin- stimulated platelets. gamma-Thrombin stimulated 80% as much fibronectin binding to platelets as alpha-thrombin with conversion of less than or equal to 1% of platelet fibrinogen to fibrin. Afibrinogenemic and normal platelets bound similar quantities of fibronectin in the presence of calcium or magnesium-ethylene glycol tetra-acetic acid (EGTA). These observations indicate that fibronectin can interact with platelets without involvement of fibrin or fibrinogen. Nevertheless, two different effects of fibrin(ogen) on fibronectin binding were observed. First, exogenous fibrinogen inhibited fibronectin binding to thrombin-stimulated platelets. This inhibition was unidirectional, as fibronectin did not inhibit fibrinogen binding to ADP or thrombin- stimulated cells. Second, formaldehyde-fixed cells with surface- associated fibrin bound significant quantities of fibronectin. This interaction required calcium and did not occur on fixed cells with or without surface-bound fibrinogen. A portion of the ligand bound to fixed cells with surface-associated fibrin was modified to form a derivative with a molecular weight identical to that of the fibronectin subunit cross-linked to the alpha-chain of fibrin. This high mol wt derivative was also observed to a variable extent with living cells in the presence of magnesium or calcium but not in the presence of magnesium-EGTA. Thus, fibronectin binds to platelets by at least two mechanisms: (1) a fibrin(ogen)-independent pathway that requires divalent ions and is inhibited by exogenous fibrinogen; and (2) a fibrin-dependent pathway with an absolute calcium requirement. With nonaggregated, thrombin-stimulated platelets, the former pathway appears to predominate.     

Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets.

We have constructed synthetic peptides modeled on the sequences of (i) Arg-Gly-Asp, present in fibrinogen, fibronectin, and von Willebrand factor, and of (ii) the fibrinogen gamma chain (gamma 400-411) His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val. The concentration of each peptide that inhibits 50% of 125I-labeled fibrinogen binding to thrombin-stimulated platelets (IC50) was then determined. The IC50 for (gamma 400-411) was 48-180 microM at a fibrinogen concentration of 60 micrograms/ml. A substitution of arginine for alanine at position 9 decreased the IC50 to 14.5 microM. Arginine substitutions for all other residues on the amino-terminal side of the peptide Arg9-Gly-Asp-Val resulted in an IC50 of 0.4-0.8 microM, and the IC50 of the peptide Arg13-Gly-Asp-Val was 0.2-0.3 microM. This contrasts with an IC50 of 200 microM for Arg5-Gly-Asp-Val-Arg4 and an IC50 greater than 1 mM for the peptide Arg12. The inhibitory effect resulted primarily in a decreased affinity of fibrinogen binding to platelets, although the number of available binding sites had also decreased. Binding was completely inhibited. At concentrations between 10 and 18 microM, Arg9-Gly-Asp-Val blocked all ADP-induced aggregation in citrated platelet-rich plasma. The peptide Tyr-His-His-Lys-Arg-Lys-Arg-Lys-Gln-Arg-Gly-Asp-Val was labeled with 125I to quantitate its binding to thrombin-stimulated platelets; at saturation, 59,990 molecules were bound per cell (Kd = 3.8 X 10(-7) M). These modified synthetic peptides bind to platelets with the same affinity as does intact fibrinogen and inhibit platelet function. The increased affinity of these modified peptides is greater than 20-fold that of peptides comprised of only native sequences and is a prerequisite for the potential antithrombotic use of these agents.     

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet-fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.     

Effect of epinephrine on fibrinogen receptor exposure by aspirin-treated platelets and platelets from concentrates in response to ADP and thrombin

Synergistic effects between agonists on platelet aggregation have long been appreciated. Recently epinephrine was reported to induce maximal aggregation of aspirin-treated platelets when combined with ADP or thrombin, and to increase fibrinogen binding of non-aspirin treated platelets stimulated with low doses of ADP. The present study extends these observations to correlate fibrinogen binding in response to various combinations of ADP, epinephrine, and thrombin with platelet aggregation and 14C-serotonin release using aspirin-treated platelets as well as platelets from stored concentrates. When fresh platelets were stimulated with epinephrine (5 microM) together with either ADP (10 microM) or thrombin (150 mU/ml), fibrinogen binding increased by 180% compared to binding observed in response to ADP or thrombin alone. This was accompanied by enhanced platelet aggregation, but no increase in 14C-serotonin release. While both ADP and epinephrine potentiated the aggregation and fibrinogen binding of stored platelets in response to high doses of thrombin (150 mU/ml), maximal aggregation was achieved only with thrombin (150 mU/ml) and epinephrine (5 microM) in combination. The data thus suggest that 1) epinephrine induces maximal aggregation of aspirin-treated platelets stimulated with thrombin or ADP by significantly enhancing fibrinogen receptor exposure independently of the cyclooxygenase-mediated release reaction; 2) epinephrine stimulates platelets by a mechanism different from that of thrombin or ADP; and 3) as demonstrated by others, the ability of platelets from stored concentrates to aggregate and to bind fibrinogen in response to ADP can be enhanced by epinephrine, and, in addition, these platelets can aggregate and bind fibrinogen maximally when stimulated with combinations of epinephrine and thrombin.     

The mitosis of fibroblasts in cell culture is enhanced by binding GP IIb-IIIa of activated platelets on fibrinogen

A fibroblast cell culture model enables us to measure the mitogenic ability mediated by growth factors released from stimulated platelets under different conditions. Simultaneously the growth factors secreted in the culture medium were determined. Cell mitotic rate was measured by incorporation of 3H-thymidine on days 3, 5 and 7 of culture. PDGF, TGF-beta, EGF and IGF-I were determined by Western blot. When fibroblasts were grown on surfaces precoated with a mixture of fibrinogen and thrombin-stimulated platelets, the 3H-thymidine uptake (196,645 +/- 56,864 cpm/ml) was increased, in comparison to fibroblasts grown on uncoated surfaces, in medium supplemented with FBS (28,855 +/- 7329 cpm/ml). Neither thrombin-stimulated platelets without fibrinogen nor fibrinogen alone had positive effects on the mitogenic activity of fibroblast. Growth factors were identified only in a culture medium in which the cells were grown on surfaces precoated with fibrinogen and thrombin-stimulated platelets. Blocking the platelet integrin GP IIb-IIIa inhibited the release of growth factors from thrombin-stimulated platelets and consecutively the stimulation of mitosis by fibrinogen and activated platelets was absent. Antibodies against the growth factors added to the medium suppressed the stimulation of cell mitosis. These results show that delivery of growth factors from platelets' secretory granules is dependent on binding of fibrinogen to GP IIb-IIIa.     

Correlation between fibrinogen binding to human platelets and platelet aggregability

Fibrinogen is essential for aggregating platelets with adenosine diphosphate (ADP) and was recently shown to bind to platelets stimulated with ADP. The present work confirms the specific and saturable nature of the platelet-fibrinogen interaction. Binding of 125iodine-labeled fibrinogen to human gel-filtered platelts was maximal at 1 min, and the receptors were saturated when the fibrinogen concentration in the suspending medium approached 0.8 mg/ml. Assuming that one fibrinogen molecule interacts with a single receptor, experiments with 9 normal donors revealed the presence of 12,896 +/- 2456 receptors per platelet. Much of the bound material dissociated from platelets after incubation with apyrase or EDTA. Binding was markedly inhibited at pH 6.5, in the presence of EDTA, and with platelets from 3 thrombasthenic patients but not with those from a patient with the Bernard-Soulier syndrome. Fibrinogen binding was also virtually absent with platelets that had been incubated with EDTA for 8 min at 37 degrees C and pH 7.8. These platelets could not aggregate when mixed with ADP and adequate CaCl2 and fibrinogen, although they could still change their shape. Thus, ADP-induced binding of fibrinogen correlates with platelet aggregability.     

Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane

Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]- prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb- IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.