The hemostatic effect of bovine peptide-B from fibrinogen

Bovine peptide-B from fibrinogen was active in the hemostasis of rat tail arterioles. The time of bleeding exhibits an inverse log proportionality to the concentration of bovine peptide-B. The peptide produced a 10-fold decrease in the time of bleeding at the highest concentration tested. Prolonged incubation of the peptide with the system was unnecessary and it appeared to participate immediately as a hemostatic agent. These findings suggest that hemostasis was due to vasoconstriction since coagulation time remained constant as the bleeding time decreased with increasing concentration of peptide-B. Bovine peptide-B is interpreted as a physiological substance which probably acts on some smooth muscle receptor.     

Human platelet basic protein associated with antiheparin and mitogenic activities: purification and partial characterization.

Platelet basic protein (PBP) (isoelectric point, 10.0-10.5; apparent Mr, 11,000-15,000) has been purified to homogeneity from material secreted by fresh human platelets after stimulation by thrombin. The purification, using preparative isoelectric focusing and chromatography on heparin-Sepharose, yielded two additional peptides with antiheparin activity that were immunologically identical with PBP: low-affinity platelet factor 4 and beta-thromboglubulin. The purity of the peptides was confirmed by immuoelectrophoresis and by NH2-terminal amino acid analysis. Dansyl chloride-treated PBP yielded a single dansylated amino acid residue (glycine). By using a specific radioimmunoassay it was shown that 10(9) human platelets contain 2-3 microgram of PBP which can be released in response to specific stimulation. PBP is associated with mitogenic activity as assayed in Swiss 3T3 mouse cells cultured in low-serum (0.4-1.5%) medium at levels of about 1 ng/ml and saturating at 10-40 ng/ml. The biological activity of different PBP preparations was variable, presumably due to inhibition by the varying amounts of ampholytes that interfered with the mitogenic activity of the peptide. Mitogenic activity was eluted from NaDodSO4/polyacrylamide gels and shown to comigrate with immunoreactive material and with conventional marker proteins of 14,000-17,000 daltons or with histones of 11,000-15,000 daltons. Evidence is presented that PBP is different from cationic platelet-derived growth factor which has an apparent Mr of 30,000.     

ADP and epinephrine-induced release of platelet fibrinogen

Human platelets gel-filtered into Tyrode's buffer containing 1 mM Mg++ and 0.35% bovine serum albumin were studied to determine whether they would undergo biphasic aggregation and release of alpha-granule proteins in response to adenosine diphosphate (ADP) or epinephrine without addition of exogenous fibrinogen. Fibrinogen concentration in the supernatant of unaggregated gel-filtered platelets was less than 1 pmole/ml. With addition of ADP or epinephrine, biphasic aggregation was seen, with release of platelet fibrinogen, beta-thromboglobulin, and platelet factor 4. Fibrinogen concentration in the supernatant after aggregation ranged from 15 to 70 pmole/ml. Release of the alpha-granule proteins by epinephrine was coincidental with release of the dense granule adenine nucleotides. Aggregation and alpha-granule protein release by both ADP and epinephrine were inhibited by added Ca++ at 1-- 2 mM. The ability of gel-filtered platelets to undergo ADP- and epinephrine-induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen may be able to fulfill the requirement for fibrinogen in ADP- and epinephrine-induced platelet aggregation and release.     

Common structural genes for platelet and plasma fibrinogen

Fibrinogen from plasma was compared with fibrinogen from platelets using two-dimensional electrophoresis. The source of platelet fibrinogen was isolated alpha-granules, thrombin- and collagen-released platelet material. The B beta- and gamma-chains from the different sources showed similar two-dimensional patterns, while gamma'-chains were not observed in platelet fibrinogen preparations. Furthermore, the A alpha-chain could hardly be identified in platelet preparations. When individual fibrinogen was studied in persons heterozygous for genetic B beta- and gamma-chain variants, the two-dimensional variant pattern could be demonstrated in plasma fibrinogen as well as in platelet fibrinogen. This observation strongly indicates that the structural genes for plasma and platelet fibrinogen B beta- and gamma-chains are identical.     

Relationship of fibrinogen levels and hemostatic abnormalities with organ damage in hypertension

Elevated plasma levels of fibrinogen and activated coagulation pathways are risk factors of cardiovascular disease in the general population. In a cross-sectional study of a case series, we investigated the relationship between fibrinogen and hemostatic markers with target-organ damage (TOD) in patients with arterial hypertension. Prothrombin time, partial thromboplastin time, fibrinogen, fibrin D-dimer, prothrombin fragment 1+2 (F1+2), and antithrombin III were measured in 352 untreated patients with mild to moderate essential hypertension and 92 normotensive controls. Staging of TOD was assessed according to W.H.O. guidelines by clinical evaluation and laboratory tests including measurements of creatinine clearance, proteinuria, ophthalmoscopy, electrocardiography, echocardiography, and ultrasound examination of major arteries. F1+2 concentrations were significantly greater in hypertensive patients than normotensive controls and were positively correlated with blood pressure. Age, blood pressure levels, duration of hypertension, smoking, HDL-cholesterol, triglycerides, and plasma fibrinogen, fibrin D-dimer, and F1+2 levels were significantly related to the presence and severity of TOD in univariate analysis. Plasma fibrinogen and D-dimer levels were related to organ damage independent of age, blood pressure, duration of hypertension, and smoking status. Separate analysis indicated significant association of fibrinogen and D-dimer levels with cardiac, cerebrovascular, peripheral vascular, and renal damage. In conclusion, elevated plasma levels of fibrinogen and a prothrombotic state are associated with the presence and severity of TOD in patients with essential hypertension and may contribute to the development of atherosclerotic disease in these patients.     

Evidence for interaction between platelet fibrinogen receptors

Previous analysis of fibrinogen binding to human aspirin-treated gel- filtered platelets yielded upwardly concave Scatchard plots. To ascertain whether this was due to the presence of independent heterogeneous receptor populations binding fibrinogen with different affinities, the dissociation of purified 125I-fibrinogen from ADP- treated gel-filtered platelets was evaluated as a function of receptor occupancy. Dissociation of bound labeled fibrinogen was measured after 50-fold dilution with buffer containing O, 0.2, 0.8, and 2.0 mg/ml unlabeled fibrinogen. Dissociation of labeled fibrinogen increased with increasing receptor occupancy and was biphasic. With buffer alone, 76.0% +/- 5.8% (SD) of labeled fibrinogen dissociated in 30 min, with an initial rate of 0.392 +/- 0.175 min-1; with 0.2 mg/ml fibrinogen, 83.7% +/- 3.9% dissociated, with an initial rate of 0.589 +/- 0.044 min- 1; with 0.8 mg/ml, 91.8% +/- 1.3% of the labeled fibrinogen dissociated, with an initial rate of 0.910 +/- 0.028 min-1; and with 2.0 mg/ml fibrinogen, 97.3% +/- 2.3% of label dissociated, with an initial rate of 1.06 +/- 0.257 min-1 (n=5). The final rates of fibrinogen dissociation were unaffected by unlabeled fibrinogen in the dilution buffer and were not statistically different from the final dissociation rate of 0.015 +/- 0.10 min-1 observed following dilution with buffer alone. These results were neither an artifact of aspirin treatment or gel filtration, as similar observations were made using non-aspirin-treated washed platelets, nor were they an artifact of the purified fibrinogen preparations, because binding studies using whole plasma as the major source of fibrinogen also yielded upwardly concave Scatchard plots. Since the data demonstrate that the initial rate and extent of fibrinogen dissociation are dependent on fibrinogen receptor occupancy, they suggest receptor interactions possibly resulting from receptor clustering or crosslinking. Because the dissociation was biphasic, the results also suggest some heterogeneity among platelet- fibrinogen interactions.     

Internalization of bound fibrinogen modulates platelet aggregation

In agonist-stimulated platelets, the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) is converted from an inactive to an active fibrinogen receptor, thereby mediating platelet aggregation. With time after agonist addition, at least two events occur: fibrinogen becomes irreversibly bound to the platelet and, when stirring is delayed, platelets lose the ability to aggregate despite the presence of maximally bound fibrinogen. Because we previously identified an actively internalized pool of alpha IIb, beta 3 in platelets, we explored the possibility that both of these events might result from the internalization of fibrinogen bound to active alpha IIb beta 3. Under conditions of irreversible fibrinogen binding, fluorescence microscopy showed that biotinylated fibrinogen is rapidly internalized by activated platelets to a surface-inaccessible, intracellular pool. Flow cytometric analysis showed that the observed loss in accessibility to extracellular probes immediately precedes a loss in ability to the platelets to aggregate. Moreover, prevention of irreversible fibrinogen binding results in a prevention of internalization and a retention of aggregation capacity. Thus, the internalization of fibrinogen from the activated platelet surface appears to contribute not only to the irreversible phase of fibrinogen binding, but also to the downregulation of platelet adhesiveness. Fibrinogen internalization is therefore likely to represent a fundamental regulatory mechanism that modulates platelet function.     

Human heart citrate synthase: purification, properties, kinetic and immunologic studies.

Citrate synthase from human heart was purified by affinity chromatography with Sepharose-ATP. The molecular weight (100 000) and presence of two presumably identical subunits do not differ from other mammalian citrate synthases. However, the kinetic constants, and immunologic characteristics of the enzyme, differed from findings from other mammalian citrate synthases. The K_m values for acetyl CoA (0.4 μm) and oxaloacetate (0.25 μm) were about an order of magnitude lower than that previously found for other mammalian (and eucaryotic) citrate synthases. The kinetic constants for the reverse reaction, K_m for citrate (420 μm) and CoA (70 μm) were of similar magnitude to the values for other mammals. Anti-human heart antiserum developed a single precipitin line in an Ouchterlony plate against a heart extract, no precipitin line with brain, and a precipitin line with spurs against liver and kidney extract. Following myocardial infarction in men, the enzyme appeared in peripheral blood rarely and in low concentration in contrast with earlier experiences with experimental infarction in dogs.     

Flow cytometric analysis of platelet activation and fibrinogen binding

Human blood platelets can be activated by a variety of physiological activators such as adenosine diphosphate (ADP), thrombin or collagen, leading to activation of GPIIb-IIIa into a high-affinity receptor for Fg (FgR), binding of fibrinogen (Fg), and subsequent platelet aggregation required for normal hemostasis. Although enormous progress has been made in the biochemistry of platelet activation, of the platelet membrane GPIIb-IIIa, and of solution Fg, much less is known of the dynamics of expression of FgR, of its occupancy by Fg, and of their relation to the dynamics of platelet aggregation. Since platelet activation and aggregation occur within ～1 s of stirring with activators such as ADP, a methodology was required for determining the rapid dynamics of expression of FgR and binding of Fg, and their correlation with platelet aggregation kinetics. We therefore developed the theoretical and experimental base for determining these dynamic changes under non-equilibrium conditions, using fluorescently-labelled probes and flow cytometry. This approach has yielded a novel general technique for assessing the rapid dynamics of any cell surface molecule, as well as unexpected new insights into the kinetic expression and nature of FgR formed on platelet surfaces activated with ADP and PMA. The same approach has been extended to an analysis of the size-dependent (subpopulation) behaviour of platelets in expressing FgR, obtainable by analytically selecting platelets of different size from forward scatter profiles obtained in studies of the whole population. Parallel measurements of kinetics of platelet recruitment into microaggregates and expression and Fg occupancy of FgR as a function of ADP concentration, led to an unexpected new model for platelet activation and recruitment based once again on the selective recruitment of platelet subpopulation and an ‘all or none, quantal’ response of any single platelet in expressing all of its FgR and becoming recruitable for aggregation, but at a critical ADP concentration dependent on its own subpopulation characteristics. This approach has also led to novel insights into problems associated with platelet ‘activation’ arising with different isolation procedures. Dynamic binding studies of Fg to FgR on activated platelets has become possible using appropriately FITC-labelled Fg and flow cytometry. This has also led to studies of the relation between shear-dependent capture efficiency of platelets into doublet formation and the fraction of Fg-occupied receptors. In addition, we have successfully used FITC-labelled human and bovine Fg to demonstrate a delayed expression of FgR and Fg binding to ADP-activated platelets from bleeding Simmental cattle, although the final expression of numbers and accessibility of FgR, measured at equilibrium, were normal. Some future directions for dynamic flow cytometric studies of platelet activation and function are discussed.     

Flow cytometric analysis of platelet activation and fibrinogen binding

Human blood platelets can be activated by a variety of physiological activators such as adenosine diphosphate (ADP), thrombin or collagen, leading to activation of GPIIb-IIIa into a high-affinity receptor for Fg (FgR), binding of fibrinogen (Fg), and subsequent platelet aggregation required for normal hemostasis. Although enormous progress has been made in the biochemistry of platelet activation, of the platelet membrane GPIIb-IIIa, and of solution Fg, much less is known of the dynamics of expression of FgR, of its occupancy by Fg, and of their relation to the dynamics of platelet aggregation. Since platelet activation and aggregation occur within ～1 s of stirring with activators such as ADP, a methodology was required for determining the rapid dynamics of expression of FgR and binding of Fg, and their correlation with platelet aggregation kinetics. We therefore developed the theoretical and experimental base for determining these dynamic changes under non-equilibrium conditions, using fluorescently-labelled probes and flow cytometry. This approach has yielded a novel general technique for assessing the rapid dynamics of any cell surface molecule, as well as unexpected new insights into the kinetic expression and nature of FgR formed on platelet surfaces activated with ADP and PMA. The same approach has been extended to an analysis of the size-dependent (subpopulation) behaviour of platelets in expressing FgR, obtainable by analytically selecting platelets of different size from forward scatter profiles obtained in studies of the whole population. Parallel measurements of kinetics of platelet recruitment into microaggregates and expression and Fg occupancy of FgR as a function of ADP concentration, led to an unexpected new model for platelet activation and recruitment based once again on the selective recruitment of platelet subpopulation and an 'all or none, quantal' response of any single platelet in expressing all of its FgR and becoming recruitable for aggregation, but at a critical ADP concentration dependent on its own subpopulation characteristics. This approach has also led to novel insights into problems associated with platelet 'activation' arising with different isolation procedures. Dynamic binding studies of Fg to FgR on activated platelets has become possible using appropriately FITC-labelled Fg and flow cytometry. This has also led to studies of the relation between shear-dependent capture efficiency of platelets into doublet formation and the fraction of Fg-occupied receptors. In addition, we have successfully used FITC-labelled human and bovine Fg to demonstrate a delayed expression of FgR and Fg binding to ADP-activated platelets from bleeding Simmental cattle, although the final expression of numbers and accessibility of FgR, measured at equilibrium, were normal. Some future directions for dynamic flow cytometric studies of platelet activation and function are discussed.     

Peroxynitrite-mediated modification of fibrinogen affects platelet aggregation and adhesion

The reaction of peroxynitrite with fibrinogen resulted in both structural modifications and altered biological properties of this glycoprotein. SDS-PAGE analysis of peroxynitrite-treated fibrinogen, performed under non-reducing conditions, showed some aggregated material on the top of the gel (5-10% of total staining bands) and the presence of nitrotyrosine. The amount of nitrotyrosine, detected by immunoassay with anti-nitrotyrosine antibodies, was dependent on peroxynitrite concentration. In comparison with native molecule, peroxynitrite-treated fibrinogen subjected to SDS-PAGE under reducing conditions revealed not only three bands corresponding to A f , B g and n chains, but the existence of additional high molecular bands probably due to the formation of dityrosine crosslinking between fibrinogen subunits. The different susceptibility in tyrosine nitration of fibrinogen subunits was also observed. The A f chain was the most intensely nitrated, while B g and n chains were nitrated much less? Peroxynitrite-treated fibrinogen in comparison with native molecule had a distinct capability to mediate platelet adhesion and aggregation. Both unstimulated and ADP-activated platelets showed a reduced ability to adhere to peroxynitrite-modified fibrinogen. The percentage of ADP-induced platelet aggregation decreased as a function of peroxynitrite-mediated modification of fibrinogen molecule.     

Peroxynitrite-mediated modification of fibrinogen affects platelet aggregation and adhesion

The reaction of peroxynitrite with fibrinogen resulted in both structural modifications and altered biological properties of this glycoprotein. SDS-PAGE analysis of peroxynitrite-treated fibrinogen, performed under non-reducing conditions, showed some aggregated material on the top of the gel (5-10% of total staining bands) and the presence of nitrotyrosine. The amount of nitrotyrosine, detected by immunoassay with anti-nitrotyrosine antibodies, was dependent on peroxynitrite concentration. In comparison with native molecule, peroxynitrite-treated fibrinogen subjected to SDS-PAGE under reducing conditions revealed not only three bands corresponding to Aalpha, Bbeta and gamma chains, but the existence of additional high molecular bands probably due to the formation of dityrosine crosslinking between fibrinogen subunits. The different susceptibility in tyrosine nitration of fibrinogen subunits was also observed. The Aalpha chain was the most intensely nitrated, while Bbeta and gamma chains were nitrated much less? Peroxynitrite-treated fibrinogen in comparison with native molecule had a distinct capability to mediate platelet adhesion and aggregation. Both unstimulated and ADP-activated platelets showed a reduced ability to adhere to peroxynitrite-modified fibrinogen. The percentage of ADP-induced platelet aggregation decreased as a function of peroxynitrite-mediated modification of fibrinogen molecule.     

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.