The tumor-promoter phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate), a potent aggregating agent for blood platelets.

The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate, a potent tumor-promoting agent, caused irreversible platelet aggregation when more than 0.02 microM was stirred with human citrated or heparinized platelet-rich plasma (PRP). With washed platelets, 1 nM was effective. The alcohol phorbol, which has little tumor-promoting activity, failed to cause platelet aggregation. With all but low concentrations of phorbol ester, aggregation was succeeded by a rapid phase. The latter was prevented or reduced by enzymes which destroy ADP and by aspirin, was associated with a change in platelet shape, and was presumably due to released ADP. At higher concentrations, only a rapid phase was seen, and these inhibitors were not effective. Low concentrations did not aggregate platelets in PRP containing sufficient EDTA or EGTA to chelate ionized calcium or in PRP from thrombasthenic patients; higher concentrations caused slight aggregation. Both the primary, non-ADP-dependent aggregation and the rapid ADP-dependent aggregation were markedly inhibited by substances which increase cyclic AMP, metabolic inhibitors, and the sulfhydryl inhibitor N-ethylmaleimide. Phorbol ester reduced platelet cyclic AMP only when it had been previously elevated by prostaglandin E(1). 1 microM did not release beta-glucuronidase, lactic dehydrogenase, or inflammatory material from platelets in 4-5 min despite marked aggregation, but liberated all three in 30 min. The possibility is discussed that low phorbol ester concentrations cause primary aggregation by a direct action on platelet actomyosin.

Relationship of ADP-induced fibrinogen binding to platelet shape change and aggregation elucidated by use of colchicine and cytochalasin B.

ADP causes human, aspirin-treated, gel-filtered platelets to change from their native discoid shape to spiny spheres with pseudopods, bind 125I-labeled fibrinogen, and aggregate if shaken with sufficient fibrinogen. After destruction of the added ADP with the enzyme apyrase, the platelets revert to a disc shape and lose much of their bound fibrinogen. Colchicine (208 muM or 83 microgram/ml) added to ADP-treated platelets before apyrase prevented restoration of the discoid shape but not the loss of bound fibrinogen. It did not inhibit ADP-induced shape change, aggregation, or fibrinogen binding. Cytochalasin B (0.02--0.2 muM or 0.01--0.10 microgram/ml) prevented ADP-induced shape change but not ADP-induced fibrinogen binding or aggregation. Thus, these findings support earlier studies with thrombasthenic and EDTA-treated platelets and with normal platelets at low pH, or in the presence of EDTA to indicate that fibrinogen binding is associated with aggregability but not with platelet shape.

Platelet aggregation caused by dithiothreitol.

MacIntyre et al. showed that over 1 mM dithiothreitol (DTT) aggregates blood platelets in the presence of fibrinogen; aggregation is not inhibited by prostaglandin E1. We confirmed their data and found that 70 mM 2-mercaptoethanol was also active. DDT-induced aggregation was not associated with platelet shape change or secretion of dense granule contents, was not inhibited by tetracaine or metabolic inhibitors, was prevented at pH 6.5, and prevented, reversed, or arrested by EDTA, depending on when the EDTA was added. DTT did not cause aggregation of thrombasthenic, EDTA-treated, or cold (0 degree C) platelets, which also failed to aggregate with ADP. Platelets stimulated with DTT bound 125I-labeled fibrinogen. Thus DTT appears to "expose" the fibrinogen receptors. SDS gel electrophoresis of platelet fractions prepared by use of Triton X-114 showed that aggregating concentrations of DTT reduced proteins of apparent Mr 69,000 and 52,000 (probably platelet albumin) and, to a variable extent, glycoproteins Ib, IIb and III. Exposure of unlabeled or 125I-labeled platelets to ADP had no discernible effect on the electrophoretic patterns.

Modification of platelet functions by monobromobimane, a fluorescent thiol group label.

Monobromobimane (mBBr, bimane), a compound that penetrates cells and forms a fluorescent adduct with thiol groups, was used to asses the significance of thiols in platelet function. Exposure of washed platelets for 1 min to 100 microM mBBr abolished ADP-induced aggregation; shape change was not inhibited by 500 microM mBBr. The nonpenetrating compound monobromotrimethylammoniobimane was ineffective. Established ADP-induced aggregation was reversed by bimane, and fibrinogen binding to ADP-stimulated platelets was inhibited, an effect mainly due to decreased number of binding sites. Aggregation stimulated by A23187 and arachidonate was less effectively inhibited whereas epinephrine- and collagen-induced aggregation were abolished by 50 microM mBBr. Similar effects on aggregation and secretion were observed in platelet-rich plasma except that higher mBBr concentrations were usually necessary. Aggregation and 14C-serotonin secretion stimulated by 0.1 U/ml thrombin were partially inhibited by pretreatment with bimane. With lower thrombin concentrations, they were often enhanced, as was 3H-arachidonate release. Bimane inhibited epinephrine-induced arachidonate release in gel-filtered platelets, possibly because it abolished the primary aggregation necessary for this release. mBBr did not elevate cyclic AMP but enhanced the increase induced by PGE1 and prevented the subsequent decrease typically caused by ADP. Examination of SDS polyacrylamide gels with ultraviolet light showed that mBBr reacted with many platelet proteins but not with GP IIb or IIIa. This observation, and the fact that bimane did not inhibit the fibrinogen-induced aggregation of DTT- or chymotrypsin-treated platelets suggest that it reacts with thiol group(s) that are involved in "exposing" the fibrinogen receptor.

Non-decalcified barium sulfate-adsorbed plasma. A potentially useful reagent for studying blood clotting, platelets or complement.

Plasma was prepared by rapid centrifugation of native blood followed by adsorption of vitamin K-dependent clotting factors with BaSO4. Compared to fresh citrated plasma, BaSO4-treated plasma contained about 80% of factor V and factor VII and the same concentration of fibrinopeptide A. Assays were also carried out after overnight incubation of citrated plasma and BaSO4-adsorbed plasma with and without added citrate and compared to assays of fresh citrated plasma. Factor V decreased to about 25% in both citrated samples, factor VIII decreased to 45% in both samples of BaSO4-treated plasma, and fibrinopeptide A did not change. Thus loss of factor V activity depended on reduction in divalent cation concentration whereas loss of factor VIII activity may have resulted from effects of early traces of thrombin.

Evidence that von Willebrand factor is not required for the clotting of plasma in the presence of platelets and kaolin (Hardisty-Hutton test).

Antihemophilic factor (AHF) essentially free of von Willebrand factor (vWF) was used to determine whether vWF affected the coagulant activity of AHF in a kaolin-activated system using platelet-rich plasma. The AHF was prepared by sequential chromatography on solid-phase polyelectrolyte (PE E-5), and on Sepharose CL-4B in the presence of a high concentration of CaCl2. Residual traces of vWF antigen were removed by affinity chromatography with immobilized anti-vWF IgG. The essentially vWF-free AHF corrected the clotting defect of plasma from three patients with severe von Willebrand's disease (vWD) as measured by the kaolin-activated clotting time in the presence of normal or vWD platelets ( Hardisty - Hutton test). Addition of hemophilic plasma as a source of vWF did not cause additional improvement, nor did a potent antibody to vWF raised in rabbits inhibit the ability of AHF to shorten the clotting time of vWD plasma. Thus we found no evidence that vWF is necessary for AHF to function in the coagulation of recalcified kaolin-activated vWF-deficient platelet-rich plasma.

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)

The combining ability of glycoproteins IIb, IIIa and Ca2+ in EDTA-treated nonaggregable platelets.

Platelets deprived of calcium and incubated at 37 degrees C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated nonaggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X-114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37 degrees C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37 degrees C.

Increased activity of vitamin K-dependent clotting factors in human hyperlipoproteinaemia - association with cholesterol and triglyceride levels.

Levels of blood coagulation factors, cholesterol and triglyceride were measured in human plasma. Prothrombin was significantly elevated in type IIa hyperlipidaemia; prothrombin and factors VII, IX and X in type IIb; and prothrombin and factors VII and IX in type V. Multiple regression analysis showed significant correlation between the levels of these plasma lipids and the vitamin K-dependent clotting factors (prothrombin, factors VII, IX and X). Higher cholesterol levels were associated with higher levels of prothrombin and factor X while higher triglyceride levels were associated with higher levels of these as well as factors VII and IX. Prothrombin showed a significant cholesterol-triglyceride interaction in that higher cholesterol levels were associated with higher prothrombin levels at all levels of triglyceride, with the most marked effects in subjects with higher triglyceride levels. Higher prothrombin levels were noted in subjects with high or moderately elevated (but not low) cholesterol levels. Ultracentrifugation of plasma in a density of 1.21 showed activity for prothrombin and factors VII and X only in the lipoprotein-free subnatant fraction. Thus, a true increase in clotting factor protein was probably present. The significance of the correlation between levels of vitamin K-dependent clotting factors and plasma lipids remains to be determined.

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.