Expression of intracellular fibrinogen on the surface of stimulated platelets.

Participation of fibrinogen in platelet aggregation is contingent upon the capacity of various stimuli to induce specific receptors for the molecule on the surface of the cell. The interaction of fibrinogen with this receptor results directly in platelet aggregation, and dissociation of fibrinogen is associated with disaggregation. While the role of exogenous fibrinogen in this process has been fully documented, the mechanisms which control the surface exposure of platelet fibrinogen are less understood. In the present study Fab fragments of antibodies monospecific for fibrinogen have been used to examine the surface expression of intracellular fibrinogen and its involvement in platelet aggregation. Radiolabelled Fab fragments did not interact with non-stimulated platelets but significant binding was observed when the cells were stimulated by ADP, thrombin, collagen and Ca ionophore A23187. Binding was specific for fibrinogen, was not observed with thrombasthenic platelets and was dependent upon the presence of extracellular calcium. With all stimuli tested, the binding of the Fab probe to platelets correlated with platelet secretion. At the following concentrations of stimuli: 30 microM ADP, 4 micrograms/ml collagen, 3 microM A23187 and 0.05 U/ml thrombin, the immune Fab fragments inhibited platelet aggregation. A monoclonal antibody to glycoprotein IIb/IIIa complex and a synthetic peptide gamma 400-411, that inhibited the interaction of plasma fibrinogen with platelets, did not inhibit the binding of 125I-FAB fragments. Taken together these results support the hypothesis that endogenous fibrinogen becomes surface-expressed during stimulation of the cell and can support platelet aggregation, particularly that induced by low concentrations of stimuli. The mechanism for the surface expression of platelet fibrinogen may be distinct from that for the binding of plasma fibrinogen.

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.

Related binding mechanisms for fibrinogen, fibronectin, von Willebrand factor, and thrombospondin on thrombin-stimulated human platelets.

Fibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IIIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by greater than 85%. T10, an antibody specific for the GPIIb-IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.

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.

The platelet-fibrinogen interaction. Evidence for proximity of the A alpha chain of fibrinogen to platelet membrane glycoproteins IIb/III.

Fibrinogen participates in platelet aggregation via specific inducible receptors on the cell surface. We have used a photoactivable bifunctional reagent, N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate, SANAH, to derivatize 125I-labeled-fibrinogen (125I-Fg) and crosslink it to ADP-stimulated platelets. Binding experiments established that 125I-Fg and 125I-Fg-SANAH interacted with platelets with the same kinetics and affinity as unlabeled fibrinogen. After photoactivation of the platelet-bound 125I-Fg-SANAH, polyacrylamide gel electrophoresis under reducing conditions revealed formation of a high molecular weight covalent complex with coordinate loss of the A alpha chain. 125I-Fg-SANAH missing the extreme carboxy-terminal region of the A alpha chain failed to crosslink to the platelets under similar conditions. Crosslinked 125I-Fg-SANAH was extracted from the cells in 1% Triton X-100, and immunoprecipitation with antibodies specific for platelet membrane glycoproteins was used to identify components of the complex. With antibodies to the glycoprotein IIb/III complex (anti-GP IIb/III), 40 +/- 9% of the extracted 125I-Fg-SANAH was immunoprecipitated. Omission of photoactivation, platelets, or ADP from the reaction or use of unmodified 125I-Fg resulted in less than 5% immunoprecipitation by the anti-GP IIb/III. As controls for specificity, anti-(glycoprotein Ib) or anti-IgG immunoprecipitated less than 5% of the extracted 125I-Fg-SANAH. Under similar conditions, 45% of the GP IIb/III from surface-labeled platelets was recovered in the anti-GP IIb/III immunoprecipitate. These results indicate that the A alpha chain of fibrinogen comes in close proximity to GP IIb/III when the molecule is bound to its platelet receptor.

The fibrinogen-dependent pathway of platelet aggregation.

Fibrinogen participates in platelet aggregation induced by ADP and this participation is receptor mediated. The role of fibrinogen in platelet function is not restricted to the ADP stimulus as the molecule may regulate aggregation induced by the direct interaction of the protein with a single, specific receptor system. Thus, a common fibrinogen-dependent mechanism leading to platelet aggregation is hypothesized.

The binding of fibrinogen to its platelet receptor.

Specific receptors for fibrinogen can be induced on platelets by a variety of stimuli. In this study, two independent approaches have implicated the D domain of fibrinogen in its interaction with the platelet. Immunochemically purified Fab fragments of anti-fibrinogen and anti-D inhibited 125I-fibrinogen binding to platelets in a dose-dependent fashion and platelet aggregation. In contrast, Fab fragments of anti-E produced only a slight inhibition of fibrinogen binding and nonimmune Fab fragments had no effect. Fibrinogen was digested with plasmin in the presence of 5 mM calcium and fragment D and E of Mr 100,000 and 50,000, respectively, were isolated. This D fragment inhibited 125I-fibrinogen binding to platelets in a concentration-dependent fashion, whereas the E fragment was ineffective. With 125I-fibrinogen at 0.17 microM, nonlabeled fibrinogen inhibited binding by 50% at 0.7 microM, whereas 170 microM fragment D was required to produce 50% inhibition. D fragment of Mr 80,000, generated in the absence of calcium, was noninhibitory. These observations provide strong evidence for the participation of the D domain in the binding of fibrinogen by its platelet receptor and suggest that the recognition site is lost in the conversion of the Mr 100,000 to 80,000 D species.

The interaction of fibrinogen with human platelets in a plasma milieu.

Fibrinogen binds to specific receptors on human washed platelets and these sites are induced by adenosine diphosphate (ADP). This interaction is assumed to be the basis for the participation of the molecule in ADP-stimulated aggregation of platelets, but fibrinogen binding to platelets in plasma has not been directly demonstrated. In this study, we have characterized the interaction of 125I-fibrinogen to platelets in the platelet-rich plasma (PRP) of afibrinogenemic patients. In either citrated or heparinized PRP, association of fibrinogen with platelets was demonstrable and was dependent on ADP dose. This binding reached equilibrium in 10-15 min, and saturation was achieved at fibrinogen concentrations greater than 0.5 microM. A linear Scatchard plot was derived that indicated a single class of binding sites with an affinity constant of Ka = 1.8 X 10(6) M(-1), and 32,000 fibrinogen molecules were maximally bound per platelet. The kinetics of the platelet fibrinogen interaction in plasma were essentially the same at 37 degrees C and 22 degrees C, but fewer molecules were bound at 37 degrees C. The rate constants of association were k 22 degrees C on = 0.9 X 10(6) M-1 . min-1 and k 37 degrees C on = 0.4 X 10(6) M-1 . min-1, respectively. Stabilization of the platelet-bound fibrinogen occurred only to a partial extent in both heparinized and citrated plasma. These results are similar to those obtained with washed platelets and establish that the previously defined steps in ADP-induced binding of fibrinogen to platelets occur in plasma, namely receptor induction by ADP, initial reversible binding, and irreversible binding.

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.

Participation of ADP in the binding of fibrinogen to thrombin-stimulated platelets.

Thrombin and adenosine diphosphate (ADP) supported the binding of 125I-fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.