Induction of inhibitory activity for B cell differentiation in human CD8 T cells with pokeweed mitogen, dimaprit, and cAMP upregulating agents: countersuppressive effect of platelet factor 4.

As shown previously, native or recombinant (r) human platelet factor 4 (PF4) alleviates the suppression induced by Con A or dimaprit, a histamine type 2 receptor (H2-R) agonist, in a murine system. The effect of rPF4 on human peripheral blood cells has now been studied, using as a model pokeweed mitogen (PWM)-induced, T-cell-mediated suppression of Ig-secreting cell (ISC) formation by Staphylococcus aureus and rIL-2 activated B cells. PWM, but not phytohemagglutinin (PHA), induced inhibitory activity in mitomycin-treated CD8+ T cells, but not unfractionated or CD4+ T cells, for both ISC formation and B cell proliferation. rPF4 and its C-terminal tridecapeptide alleviated the suppressive effect of PWM-activated CD8+ T cells on ISC production but not on proliferation. Heparin did not prevent this immunoregulatory activity of PF4. Neutralizing antibody to TGF-beta, but not to IFN-gamma or TNF-alpha, alleviated the suppression of ISC formation in some of the experiments. The H2-R appeared to play a part in inducing suppression, because the H2-R antagonist, cimetidine, prevented the PWM-induced suppression of ISC production. Furthermore, dimaprit induced suppression of ISC formation when added instead of PWM at the start of culture. Incubation of CD8+ T cells with dimaprit for only 3 hr prior to coculture with S. aureus + IL-2 activated B cells decreased the ISC response. This suppression was also alleviated by addition of rPF4 to the coculture. Similar to dimaprit, known cAMP upregulating agents, such as forskolin, dibutyryl cAMP, and cAMP analog, all induced this immunoregulatory activity in T cells. Moreover, the effect of dimaprit was prevented by the specific protein kinase A inhibitor, HA1004, suggesting strongly that upregulation of cAMP played a role in the H2-R-mediated effect. Cell contact appeared to be necessary, since supernatants from dimaprit or PWM activated T cells failed to suppress ISC production. We suggest that the known ability of PF4 to prevent TGF-beta-mediated effects on endothelial and other target cells may be involved in the alleviating effect of PF4 on the cell-contact-dependent CD8+ T-cell-mediated B cell suppression.

Alleviation of immunosuppression in vitro by recombinant platelet factor 4.

To elucidate the mechanism by which platelet factor 4 (PF4), a secreted platelet protein, and its C-terminal peptides alleviate suppression of the antibody response in vivo, their immunoregulatory activity was studied in vitro, using cultured spleen cells from BALB/c mice primed with sheep red blood cells (SRBC). When addition of 48 h cultured concanavalin A (Con A) blasts at 5 x 10(5)/ml significantly suppressed the anti-SRBC plaque-forming cell response, suppression was alleviated in 25 of 29 experiments by 0.2 micrograms/ml recombinant (r)PF4 (6.4 nM if rPF4 is a tetramer). The effect of Con A blasts was abolished by cimetidine, a histamine type 2 (H2) antagonist. Dimaprit, an H2 agonist, at 1-2 x 10(-4) M, or splenic T cells that had been incubated for 1 h with dimaprit and washed, also caused significant suppression that was alleviated by 0.2 micrograms/ml rPF4 (n = 8), and by 0.02 micrograms/ml in six of these tests. The C-terminal 13 amino acid peptide of PF4 was active at 0.02-0.2 micrograms/ml (0.01-0.11 microM), but peptides from the middle or N-terminal end of the molecule, or IL-8, which shares structural homology with PF4, were inactive. IL-1 and IL-2 raised control responses without affecting suppression or its alleviation by rPF4. Neutralizing antibody to transforming growth factor beta 1 (TGF-beta 1) did not affect Con A blast-induced suppression and suppression induced by exogenous TGF-beta 1 (0.5 ng/ml) was not counteracted by rPF4. Blocking prostaglandin production with 0.2 or 2 microM indomethacin did not affect suppression significantly but reduced rPF4 activity; prostaglandin D2 restored the effect of rPF4.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of agonists and antagonists of platelet aggregation on von Willebrand factor-mediated platelet agglutination.

Ristocetin-induced platelet agglutination (RIPA) in EDTA-treated citrated platelet-rich plasma was reduced to 49 +/- 11% by 1.25 microM ADP, 41 +/- 14% by 1 microM A23187, and 26 +/- 7% by 0.1 microgram/ml platelet activating factor (PAF). The effect of 5-110 microM epinephrine was not dose-dependent, but varied between donors, with RIPA from 56-100% of the control. The inhibitory effects of these agonists were not altered by prior treatment of platelets with aspirin. Prior addition of 200 microM ATP (an ADP receptor antagonist acting at both high and low affinity ADP receptors) prevented the inhibitory action of ADP but not that of A23187 or PAF, suggesting that the inhibitory actions of the latter are not mediated by released ADP. As 700 microM 8-bromoadenosine 5-diphosphate (an ADP receptor antagonist acting mainly at the high affinity receptor) did not prevent ADP-induced inhibition of RIPA, interaction of ADP with the low affinity receptor is presumably responsible for its inhibitory action. As A23187, but not phorbol myristate acetate (0.1 microM) inhibited RIPA, an increase in intracellular calcium ions rather than direct stimulation of protein kinase C appears to mediate agonist-induced inhibition. Cytochalasin B (10.5-21 microM), dibucaine (0.5-1 mM), and prostaglandin E1 (25 nM), added before or after the agonist, prevented or reversed ADP-, A23187-, and PAF-induced inhibition of RIPA, suggesting that the state of the platelet cytoskeleton affects inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly and GPIIIa content of cytoskeletal core in platelets agglutinated with bovine von Willebrand factor.

The association between occupancy of the von Willebrand factor (vWf) receptor glycoprotein (GP) Ib, agglutination, and the assembly and composition of the cytoskeletal core was studied in 125I-surface-labeled aspirin-treated washed platelets. Binding of ligands to GPIIb-IIIa and platelet aggregation were abolished by addition of EDTA. Platelet agglutination induced by bovine vWf generated a complete cytoskeletal core (Triton-insoluble residue), shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be composed of actin-binding protein (ABP) (260 Kd), 235-Kd protein, myosin heavy chain (200 Kd), alpha-actinin (100 Kd), and actin (43 Kd). In addition, autoradiography of the gels showed a 125I 105-Kd GP, identified by immunoblot as GPIIIa, as well as GPIb, GPIIb, and another band at 87 Kd, probably GPIV. Neither cytoskeletal assembly nor GPIIa incorporation was altered if calpain was inhibited with leupeptin. Platelet suspensions exposed to bovine vWf without stirring (ie, nonagglutinated) or platelets in which agglutination was inhibited with ADP showed smaller cytoskeletons with little ABP, 235 Kd protein, and alpha-actinin. Autoradiographs showed mainly GPIb. Cytochalasin D (CD) and monobromobimane (MB) enhanced agglutination and prevented the inhibitory action of ADP on bovine vWf-induced platelet agglutination. CD markedly inhibited the assembly of the cytoskeletal core as well as GPIIIa retention, whereas MB resulted in a large Triton-insoluble residue which contained GPIIIa. Thus, development of a platelet cytoskeletal core is apparently not required for agglutination, but when a cytoskeletal core is assembled in agglutinated platelets, GPIIIa is retained.

Inhibition of von Willebrand factor-induced platelet agglutination by ADP does not result from reduced binding of total von Willebrand factor or its larger multimers.

Earlier experiments showed that platelet agglutination induced by von Willebrand factor (vWf) plus ristocetin was greatly diminished if adenosine diphosphate (ADP) was added first in the presence of ethylenediaminetetraacetic acid (to prevent aggregation). Platelets treated with ADP and then fixed also agglutinated less than control fixed platelets. The studies reported here demonstrate that ADP did not decrease ristocetin-induced binding of vWf whether binding was measured on suspended platelets with iodine 125-labeled vWf or on suspended or agglutinated platelets with the use of any of three 125I-labeled monoclonal antibodies that bind to vWf but that do not interfere with ristocetin-induced agglutination. Equal amounts of vWf were eluted from ristocetin/vWf-treated platelets when they were resuspended without ristocetin, whether or not the platelets had been exposed to ADP, and the vWf recovered in either case was composed only of large multimers. No evidence for an agglutination site other than glycoprotein Ib could be demonstrated by measuring agglutination of a mixture of platelets fixed after inhibition with antibody against glycoprotein Ib and platelets fixed after inhibition with ADP. We conclude that inhibition of agglutination by ADP must involve the way in which vWf is bound, because it does not result from a decreased amount or from a difference in multimer size of bound vWf.

Immunoregulatory activity of peptides related to platelet factor 4.

Platelet factor 4 (PF4), a secreted platelet protein, alleviates concanavalin A-induced immunosuppression in mice. We now find that activity also resides in (i) the C-terminal tridecapeptide of PF4 (P13S), (ii) an analog of this in which arginine replaces the lysine residues and in which the last two amino acids are absent, (iii) the C-terminal 18 amino acids of low-affinity platelet factor 4, which is very similar to P13S, and (iv) peptide fragments of P13S that contain only 5-9 amino acids. P13S treated with fluorescamine to derivatize the free amino groups retained immunoregulatory activity but did not bind to heparin-agarose. The N-terminal and middle portions of PF4, polylysine, protamine, and three unrelated peptides were inactive in this assay.

Effect of the thiol group inhibitor monobromobimane and other inhibitors on the composition of the platelet cytoskeletal core and its association with glycoprotein IIIa.

SDS-polyacrylamide gel electrophoresis was used to study the effects of the thiol inhibitor monobromobimane (MB), EDTA, and prostaglandin E1 (PGE1) on the formation and composition of the platelet cytoskeletal core (Triton-insoluble residue) and its association with glycoprotein (GP) IIIa. Stimulation or aggregation of platelets in response to ADP or thrombin increased the amount of Triton-insoluble myosin. Aggregation resulted in incorporation of [125I]GP IIIa and a new band at about 210 kDa into the cytoskeletal core. EDTA and PGE1 caused little disaggregation of platelets that were aggregated in PRP with ADP and that had secreted the contents of their granules. In contrast to EDTA, PGE1 decreased the amount of Triton-insoluble residue and its association with GP IIIa. MB added after ADP-induced aggregation caused an increase in the amount of cytoskeletal core despite marked disaggregation and a substantial decrease in core-associated GP IIIa. With aspirin-treated platelets that had not secreted, EDTA, PGE1, and MB all caused disaggregation and loss of cytoskeletal GP IIIa. MB diminished, but did not reverse, thrombin-induced aggregation of washed platelets and arrested GP IIIa incorporation into the cytoskeletal core. Concanavalin A (Con A) cross-links glycoproteins on a single platelet and induces incorporation of GP IIIa into the Triton-insoluble residue in the absence of platelet aggregation. This induction was not inhibited by MB, although this reagent, as well as aspirin, inhibited Con A-induced secretion. Since GP IIIa incorporation caused by ADP-induced aggregation differs from that caused by Con A in its susceptibility to MB, it seems unlikely that thiol groups are directly involved in the association of GP IIIa with the cytoskeletal core.

Erythrocyte aggregation in iohexol and other nonionic media.

Erythrocytes aggregate when blood comes into contact with solutions of nonionic substances such as glucose or the contrast medium iohexol, especially at a reduced pH (eg, 6.8). The aggregates are not clots because they form rapidly even in heparinized blood, do not contain fibrin, and disappear at once when they are added to plasma or other ionic media. Formation of these aggregates in solutions of glucose or iohexol can be decreased by including a low concentration of an ionic solute such as NaCl or by maintaining the pH close to 7.4.

Prevention by a platelet-derived factor (platelet factor 4) of induction of low dose tolerance to pneumococcal polysaccharides.

It was previously shown that human or mouse serum, and platelet factor 4 (PF4) prepared from human platelet releasate, counteracts nonspecific immunosuppression induced in mice by injection of concanavalin A or syngeneic gamma-irradiated lymphoma cells. The present studies show that PF4 prepared from normal mouse or human serum by absorption to heparin-agarose and elution between 0.5 and 1.5 M NaCl is also active in this respect. The ability of PF4 to counteract antigen-specific suppression of the antibody response to pneumococcal polysaccharide (pps) was now studied. PF4 derived from human or mouse serum as well as recombinant PF4 interferes with induction of antigen-specific low dose tolerance when they are injected at the same time as a low dose (0.2 microgram) of type 14 pps 3 days before an optimal immunizing dose (25 micrograms). Furthermore, injection of platelet releasate at the time of an optimal primary immunizing dose of pps type 14 enhances the secondary response to killed bacteria injected 2 weeks later, but not the primary response itself. Both effects are interpreted as due to interference with antigen-specific suppressor cell induction during primary immunization. Injection of PF4 is much less effective in reversing low dose tolerance to an optimal immunizing dose (0.1 microgram) of type 3 pps induced by injection of 0.005 microgram of this antigen. Differences in the mechanism of tolerance induction for the two pps types that might be responsible for this are discussed.

The expression in Escherichia coli of recombinant human platelet factor 4, a protein with immunoregulatory activity.

In order to establish more firmly the immunoregulatory effect of platelet factor 4 (PF4) and develop a means to provide material for possible clinical use, the nucleotide sequence for PF4 was synthesized utilizing a ligation strategy of six duplexes ranging from 27 to 43 base pairs in length. The individual oligodeoxynucleotides were synthesized on an automated system. The resultant gene segment (226 base pairs), which incorporated convenient HindIII and BamHI overhangs at the 5' and 3' ends, respectively, was cloned into the pIN-III-ompA-2-expression vector in Escherichia coli, affording a fusion protein of Mr = 8900 with 7 additional amino acids at the amino terminus and 4 at the carboxyl terminus and with aspartic acid rather than asparagine in position 47. The recombinant PF4 (rPF4) was purified by heparin-agarose affinity chromatography and reverse-phase high performance liquid chromatography. It reacted with a monoclonal mouse anti-human PF4 antibody on a Western blot and in an enzyme-linked immunosorbent assay. The rPF4 protein exhibited an immunoregulatory effect like that of human PF4 in its ability to reverse concanavalin A-induced immunosuppression in BALB/c mice.

Effect of monoclonal antibodies against von Willebrand factor and platelet glycoproteins IIb/IIIa on the platelet retention test.

The platelet retention test provides a measure of the number of platelets retained in a column of glass beads and is one of the few in vitro platelet function tests that is abnormal in von Willebrand's disease (vWd). In a two-stage test, 1 mL of blood (designated A) was passed through the column, followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which platelet retention was measured. With normal blood as A and B, retention is very high in all 5 mL of blood B. In the first stage, platelets adhere to the glass beads; this requires fibrinogen but not von Willebrand factor (vWf). The platelet-platelet adhesion in the second stage requires vWf, is dependent on release of ADP, and fails to occur if thrombasthenic platelets are tested. Retention was normal when blood from a patient with afibrinogenemia was used as blood B. We have now used monoclonal antibodies to elucidate further the mechanism of platelet retention. Five antibodies to different epitopes on vWf essentially abolished retention in the one-stage test and in the second stage of the two-stage test, but had no effect on the first stage. Thus, the entire vWf molecule must be free of antibody to function in the platelet-platelet adhesion of the second stage of this test. Binding of the antigen-antibody complex to the platelet Fc receptor was not responsible, as Fab and F(ab')2 fragments of one of the antibodies were as effective as intact antibody, and as neither heat-aggregated IgG nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2 fragments of 6D1, an antibody to platelet GP Ib that prevents binding of vWf to platelets, also inhibited the second phase of retention. An antibody that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8) inhibited both the first and second stages of retention, whereas LJ-P5, an antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused slight inhibition of retention when normal or afibrinogenemic blood was used as blood B and was reported to cause only partial inhibition of ADP-induced platelet aggregation in this afibrinogenemic patient. The results suggest that vWf is altered during rapid passage of blood through the glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which then binds the altered vWf or fibrinogen, either of which can induce platelet aggregation (platelet-platelet adhesion) and thus retention in the column.

Binding of thrombin-activated human factor VIII to platelets.

To study association of platelets with factor VIII, the purified protein was 125I-labelled with Bolton-Hunter reagent to a specific activity of 243,000-360,000 cpm/U. Autoradiographs of SDS polyacrylamide gels revealed polypeptides of VIII at Mr about 240 kDa, 90 kDa and intermediate values, as well as some radioactive contaminants, but the light chain (Mr 78/76) seen with silver stain was not labelled. After 2.5-5-fold activation with thrombin, the higher radioactive Mr band disappeared, the band at 90 kDa became more intense, and a band appeared at about 45 kDa. The radioactivity associated with platelets, studied in the presence of haemophilic BaSO4-treated plasma, was maximal after 6-8 min and increased 3-15-fold on activation with thrombin. With activated VIII, autoradiographs of platelet pellets showed only VIIIa but results are expressed as units of unactivated VIII bound. At 0.3-0-0.7 U/ml, 10(8) platelets bound 0.0008-0.004 U VIIIa. The amount bound was not affected by the ratio of unlabelled VIII to VIII labelled in the presence of a 50-fold molar excess of unlabelled Bolton-Hunter reagent. Binding increased to 1.5 U VIIIa/10(8) platelets (about 13,600 molecules per platelet) at 140 U/ml, with no evidence of saturation. Binding was not affected by monoclonal antibodies to platelet glycoproteins IIb/IIIa or Ib, quenching the thrombin before adding platelets, or aggregating the platelets with A23187 in the presence of thrombin. Qualitatively, binding of labelled VIIIa and factor Va studied by others are similar. Binding of 125I-VIIIa to aggregated and unaggregated platelets was normal in patient M.S. whose platelets were shown by others to be deficient in their ability to bind radiolabelled factor Xa and generate coagulant activity. This difference. and the fact that platelet coagulant activity is increased by platelet activation and/or aggregation, suggest that binding of Va or VIIIa alone does not determine the assembly of active proteolytic complexes on the platelet surface.

Protease-induced immunoregulatory activity of platelet factor 4.

Intravenous injection of human or mouse serum or platelet material secreted from appropriately stimulated platelets ("releasate") together with antigen alleviates the immunosuppression in SJL/J mice induced by injection of irradiated lymphoma cells or in (CB6)F1 mice induced by injection of concanavalin A. We now report that injection of releasate from 10(6) human platelets restores plaque-forming cells to the unsuppressed number; greater amounts increase responses further. Immunoregulatory activity is released from platelets exposed to thrombin in parallel with other alpha-granule components. Heparin-agarose absorbs activity. Purified platelet factor 4 (PF4) has activity; beta-thromboglobulin and platelet-derived growth factor have little or none. Activity in serum is neutralized by goat anti-human PF4. An enzymatic step is necessary for production of immunoregulatory activity. Releasates boiled immediately after platelet aggregation with 250 nM A23187 or those produced by adding A23187 in the presence of 100 microM serine protease inhibitor (p-amidinophenyl)methanesulfonyl fluoride (APMSF) are ineffective, whereas releasates boiled or mixed with APMSF after incubation for 60 min are active. Activity is generated by incubating a mixture of heparin-absorbed releasate (as enzyme source) and heparin-agarose eluate of releasate made in the presence of APMSF (as substrate source). The enzymatic step does not alter the heparin-neutralizing activity of PF4. Apparently a secreted platelet protease converts PF4 to a form with immunoregulatory activity.