Heinrich Patscheke

Investigations on a selective non-prostanoic thromboxane antagonist, BM 13.177, in human platelets

The mode of action of BM 13.177 (4-[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a new anti-aggregating and anti-thrombotic agent, was studied in human washed platelets and citrated PRP. With ASA-treated platelets, BM 13.177 (0.1 - 100 microM) did not inhibit the shape change and the aggregation induced by ADP, serotonin, adrenaline, thrombin, or collagen. Therefore, BM 13.177 is neither an antagonist of ADP, serotonin, adrenaline, thrombin, or collagen nor a common pathway inhibitor like PGE1, or an inhibitor of the platelet interactions during aggregation. However, BM 13.177 (greater than or equal to 0.1 microM) produced a dose-dependent reduction of shape change, aggregation and release of [3H]serotonin induced by the stable PGH2 analogues U 46619 and U 44069 in ASA-treated platelets or ASA-treated citrated PRP. In untreated platelets, BM 13.177 inhibited platelet activation by U 46619 or U 44069 and by exogenous arachidonic acid or by endogenous arachidonic acid mobilized by hydrogen peroxide. Consequently, the ADP- and adrenaline-induced secondary aggregation and [3H]serotonin release in citrated PRP and the major effects of collagen were also inhibited. In washed platelets treated with 10 microM arachidonic acid or 100 microM hydrogen peroxide, the formation of TXB2 was not inhibited by 10 microM BM 13.177. However, the TXB2 formation after stimulation with 1,200 microM hydrogen peroxide was partially reduced by BM 13.177 to the same extent as by PGE1. This reduction may be due to the absence of a secondary release of arachidonic acid from phospholipids if the platelets were prevented from activation by BM 13.177 or PGE1. Arachidonic acid and hydrogen peroxide also induced the shape change, aggregation and release of washed platelets when thromboxane formation was inhibited by dazoxiben. Under these conditions, BM 13.177 was able to abolish the platelet response which was due to accumulating prostaglandin endoperoxides. These results show that BM 13.177 acts as a selective antagonist of TXA2 and prostaglandin endoperoxides. Its inhibitory effect on platelet function does not depend on an inhibition of either the primary release of arachidonic acid or the activities of cyclooxygenase or thromboxane synthetase.

Platelet‐induced neutrophil activation: platelet‐expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18

Summary. Mutual contacts and platelet‐expressed fibrinogen seem to be required for the stimulation of neutrophils by activated platelets. The β2‐integrins CDllb/CD18 and CDllc/CD18 are potential receptors for fibrinogen on neutrophils. In order to investigate whether binding of fibrinogen to these integrins is involved, monoclonal antibodies (MoAbs) and Gly‐Pro‐Arg‐Pro (GPRP) peptide that inhibits fibrinogen binding to CDllc/CD18 were checked for their effects on the interaction of activated platelets and neutrophils. The luminol‐amplified chemilumi‐nescence (CL) as a measure for the oxidative burst of neutrophils was recorded simultaneously to the platelet aggregation in mixed cell suspensions. The adhesion of platelets and neutrophils was determined microscopically. The thromboxane A2 mimetic U46619 was used as a potent platelet agonist but that does not stimulate neutrophils. Stimulation of the platelets with U46619‐induced platelet aggregation and a strong CL of neutrophils. The platelet‐induced activation of neutrophils required added fibrinogen which fibronectin or thrombospondin could not substitute for. Cytochalasin D (Cyto D) that blocks actin polymerization totally abrogated the platelet‐induced CI of neutrophils. The MoAb OKM1 against CDllb, which blocks fibrinogen binding to CDllb/CD18 as well as the MoAbs I0T16 and IOT18 directed against CDlla and CD18, respectively, had no effect. In contrast, the MoAb LeuM5 which inhibits the binding of fibrinogen to CDllc/CD18 revealed a strong inhibition. Furthermore, GPRP peptide which CDllc/CD18 recognizes on the Aa‐chain of fibrinogen also strongly inhibited the platelet‐induced CL of neutrophils, whereas control peptides such as Gly‐His‐Arg‐Pro (GHRP) or Gly‐Pro‐Gly‐Gly (GPGG) had no effect. In contrast to the platelet‐induced CL of neutrophils, Cyto D, MoAb against CD11c and GPRP peptide did not inhibit the CL induced by FMLP and PAF in pure neutrophil suspensions. They also did not affect U46619‐induced platelet aggregation. The adhesion of platelets and neutrophils was neither dependent on added fibrinogen nor inhibited by Cyto D, MoAb against CD1 lc and the GPRP‐peptide. Therefore fibrinogen and actin polymerization seem not to be required for the adhesion of neutrophils to platelets. However, the activation of neutrophils depends on the interaction of CDllc/CD18 with the Aa‐chain of platelet‐expressed fibrinogen and the contractile system of neutrophils.

The pharmacological profile of the thromboxane A2 antagonist BM 13.177. A new anti-platelet and anti-thrombotic drug

BM 13.177 (4-[2-(benzenesulfonamido)-ethyl]-phenoxyacetic acid) is a representative of a new class of sulfonamidophenylcarboxylic acids which possess platelet-inhibitory and anti-thrombotic activity and inhibits the contraction of rabbit aorta stimulated by PG endoperoxides and TXA2. BM 13.177 5 mg/kg body weight p.o. protected rabbits from arachidonate-induced sudden death and greater than or equal to 10 mg/kg dose-dependently reduced the experimental thrombus formation induced in the rabbit aorta by perivascular administration of silver nitrate. In guinea-pigs, the collagen-induced bronchoconstriction was inhibited in a dose- and time-dependent fashion. The formation of TXA2 and the TXA2-induced platelet aggregation and smooth muscle contraction are probably crucial events in these experimental models. The protective effect of BM 13.177 may, therefore, be due to the TXA2-antagonizing effect of BM 13.177, which has been conclusively demonstrated in human platelets (PATSCHEKE and STEGMEIER, Thrombosis Res., 33, 277-288 (1984). The antagonism of TXA2 is supported by the observation that BM 13.177 also specifically inhibits the contraction of isolated arterial strips from rabbits which were stimulated with the thromboxane A2 mimetic U 46619. Schild-plot with a slope close to unity suggests a competitive type of antagonism. BM 13.177 exhibited neither anti-inflammatory nor ulcer-inducing activity of cyclooxygenase inhibitors. Furthermore it did not block the TXB2 formation in spontaneously clotting blood from rabbits and did not inhibit the release of prostacyclin-like activity from rabbit aortas. The lack of toxicological effects in long-term toxicity studies in rat and dog, together with the absence of objective and subjective side effects in the first human studies have encouraged us to initiate clinical trials in order to evaluate the therapeutic benefit of this new approach in humans.

Platelet activation detected by turbidometric shape-change analysis. Differential influence of cytochalasin B and prostaglandin E1

Abstract Activation of platelets by specific stimuli is associated with changes in the light transmission of the platelet suspension. EDTA avoids aggregation of activated platelets but does not interfere with platelet activation. Three optical effects which require increasing stimulus concentrations are characterized: 1) Disc-sphere transformation of platelets (=shape change I) is reflected by a decrease in the light transmission (LT) and disappearance of the change in LT when the stirring mechanism is switched on or off. 2) Higher inducer concentrations are necessary to elicit a further decrease in LT (=shape change II) which is detected independently on stirring. 3) A final increase in LT follows the shape change with stronger stimuli and indicates secretion. 2) and 3) are also found during stimulation of spheroid platelets obtained under certain washing conditions or by pre-treatment with cold. Cytochalasin B (CB) 60 μM impairs the disc-sphere transformation. Low concentrations of CB (2 μM) strongly enhance the final increase in LT and the extrusion of serotonin from platelets stimulated by activators of the release but not by ADP, epinephrine, or diamide. Prostaglandin E 1 (PGE 1 ) inhibits the three optical effects and the release. The requirement for the stepwise increase in PGE 1 concentrations to inhibit disc-sphere transformation, shape change II, and the extrusion phase of the release reaction confirms the conclusion that the optical parameters indicate varying degrees of platelet activation.

Hyperreactivity by an enhancement of the arachidonate pathway of platelets treated with cholesterol-rich phospholipid-dispersions

Abstract Human-platelets were incubated at room temperature with washing solution (controls), cholesterol-free, or cholesterol-rich dipalmitoyl-lecithin dispersions. After washing, platelets from each of the three preparations exhibited a similar well-preserved discoid form. Only those platelets which were incubated for ≥ 120 minutes with cholesterol-rich lipid dispersions showed an increase in their reactivity to the aggregating agents epinephrine, ADP, hydrogen peroxide, arachidonic acid, thrombin, and A 23187. This effect required platelet activation high enough to evoke serotonin release. Shape change and primary aggregation were not altered by any of the pretreatments of platelets. Increased serotonin release was accompanied by an enhancement of the generation of thiobarbituric acid-chromogen. Indomethacin or acetylsalicylic acid suppressed the increase in serotonin release. This shows that the hyperreactivity depends on prostaglandin synthesis. The platelets treated with cholesterol-rich lipid dispersions can be used as an in vitro model for human platelets in various diseased states which are hyperre-active due to an enhanced prostaglandin synthesis.

Thromboxane Synthase Inhibition Potentiates Washed Platelet Activation by Endogenous and Exogenous Arachidonic Acid

The effect of the thromboxane (TX) synthase inhibitors dazoxiben and imidazole on platelet activation by endogenous and exogenous arachidonic acid (AA) was tested with human washed platelets. Dazoxiben (1-20 microM) inhibited the formation of TXB2 and markedly enhanced the shape change, aggregation, and (3H)serotonin release induced by added AA or when prostaglandin synthesis from endogenous AA was triggered by collagen, hydrogen peroxide or methyl mercury chloride (methyl-Hg). Platelet activation by hydrogen peroxide (20-1200 microM) or methyl-Hg (1-5 microM) was entirely dependent on endogenous prostaglandin (PG) synthesis since acetylsalicylic acid (ASA), indomethacin or the cyclic endoperoxide/TXA2-antagonist BM 13.177 counteracted these stimulants with and without dazoxiben. Apparently, the potentiation is due to accumulating cyclic endoperoxides which during TX synthase inhibition reach greater platelet-activating potency than TXA2. Albumin or human platelet-poor plasma inhibited the platelet activation by hydrogen peroxide and methyl-Hg and suppressed the potentiation by dazoxiben. The latter effect of albumin may result from its PGD isomerase activity which redirects the cyclic endoperoxide metabolism to the platelet-inhibitory PGD2. The results show that non-platelet factors such as albumin are necessary to prevent a potentiating effect of TX synthase inhibitors on platelet activation.

The pharmacokinetic and pharmacodynamic profiles of the thromboxane A-2 receptor blocker BM 13.177

The pharmacokinetics and pharmacodynamics of BM 13.177 were investigated in eight healthy men who received a single oral dose of 800 mg on the first day and seven equal doses in 8‐hour intervals on the second to fourth days. Pharmacodynamic effects were measured ex vivo by the testing of platelet functions such as shape change, aggregation, and [3H] serotonin release. The maximum serum concentration of 6.6 or 6.7 mg/L was achieved within 1.6 hours after the first dose and within 1.5 hours after multiple doses, respectively. Afterwards, BM 13.177 was eliminated in urine with a terminal elimination t½of 0.84 or 1.0 hours after single and multiple dosing, respectively. The inhibition of platelet function showed the same close correlation with the serum concentrations of BM 13.177 after single and after multiple doses. Apparently, BM 13.177 induces neither refractoriness to BM 13.177 nor desensitization of the platelet thromboxane receptor. Because BM 13.177 was also well tolerated without subjective or objective side effects, this drug appears to be useful in evaluating the clinical benefit of thromboxane receptor blockade.

Correlation of Activation and Aggregation of Platelets. Discrimination between Anti-Activating and Anti-Aggregating Agents

Shape change and release reaction indicate different degrees of the complex platelet response termed activation. Aggregation is a variable consequence of activation. Aggregation shows a temperature dependency opposite to that shown by the preceding shape change. Aggregation increases at lower temperature and requires, in contrast to activation, extracellular Ca2+, stirring, and at a low degree of activation the presence of fibrinogen. Aggregation can enhance activation by triggering prostaglandin endoperoxide-thromboxane synthesis. If activation reaches a high level associated with the release reaction, activation is further amplified by prostaglandin endoperoxide-thromboxane synthesis emerging independent of aggregation. These mechanisms of amplification of activation are blocked by indomethacin, an inhibitor of prostaglandin endoperoxide-thromboxane synthesis=anti-activating agent. In contrast, anti-aggregating agents, exemplified here with n-acetyl neuraminic acid, attack the aggregation of activated platelets but neither activation nor prostaglandin endoperoxide-thromboxane synthesis. Its anti-aggregating effect, in addition, enables n-acetyl neuraminic acid to imitate the inhibitory effect of indomethacin on the feedback amplification which results from aggregation. Anti-aggregating agents as characterized here may open a new valuable concept for anti-aggregation in vivo.

Current concepts for a drug-induced inhibition of formation and action of thromboxane A2.

SummaryUrinary and plasma metabolites of thromboxane A2 (TxA2) indicate an increased TxA2 synthesis in a number of diseases, whereby TxA2 is assumed to contribute to the underlying pathomechanisms by its profound effects on platelet aggregation and smooth muscle contraction. In some clinical situations the increment in TxA2 biosynthesis is accompanied by an increased formation of prostacyclin (PGI2) which is one of the most potent inhibitors of platelet activation and smooth muscle contraction. Therefore, drugs are being developed which suppress the formation or action of TxA2 without interfering with its functional antagonist PGI2.Low doses of acetylsalicylic acid (ASA) preferentially inhibit cyclooxygenase activity in platelets and the synthesis of TxA2 in vivo. However, neither low doses (approximately 300 mg/day) nor very low doses spare the formation of PGI2 completely. Despite its limited selectivity, very low dose ASA (approximately 40 mg/day) provides an attractive perspective in TxA2 pharmacology.Although thromboxane synthase inhibitors selectively suppress TxA2 biosynthesis PGH2 can accumulate instead of TxA2 and substitute for TxA2 at their common TxA2/PGH2 receptors. Thromboxane synthase inhibitors can only exert platelet-inhibiting and vasodilating effects if PGH2 rapidly isomerizes to functional antagonists like PGI2 that can be formed from platelet-derived PGH2 by the vessel wall.TxA2/PGH2 receptor antagonists provide a specific and effective approach for inhibition of TxA2. These inhibitors do not interfere with the synthesis of PGI2 and other prostanoids but prevent TxA2 and PGH2 from activating platelets and inducing smooth muscle contractions. Most of the available TxA2/PGH2 receptor antagonists produce a competitive antagonism that can be overcome by high agonist concentrations. Since in certain disease states very high local TxA2 concentrations are to be antagonized, non-competitive receptor antagonists may be of particular interest. Some recent TxA2/PGH2 receptor antagonists produce such a non-competitive type of inhibition due to their low dissociation rate constant. As a consequence, agonists like TxA2 or PGH2 only reach a hemiequilibrium state at their receptors, previously occupied by those antagonists.A combination of a thromboxane synthase inhibitor with a TxA2/PGH2 receptor antagonist presents a very high inhibitory potential that utilizes the dual activities of the synthase inhibitor to increase PGI2 formation and of the receptor antagonist to antagonize PGH2 and TxA2. Such combinations or dual inhibitors, combining both moieties in one compound, prolong the skin bleeding time to a greater extent than thromboxane synthase inhibitors and even more than low dose ASA or TxA2/PGH2 receptor antagonists.

The origin of the membrane convolute in degranulating platelets: a comparative study of normal and gray platelets

SummaryThrombin-stimulated normal platelets contain a membrane system of dilated channels with openings to the exterior. Whether these membranes originate from the surface connected system (SCS), the α-granules or internalized portions of the plasmalemma has not yet been defined. The present study traces in series of ultrathin sections the rearrangement of these membranes during shape change, degranulation and internalization of surface membranes in washed normal and “gray” platelets upon the stimulation with thrombin (1 IU/ml). Cationized ferritin (CF) was used as a surface marker in order to recognize internalized portions of the plasmalemma. Within the first seconds after stimulation, both normal and gray platelets changed their shape by extrusion of the SCS membranes. Simultaneously they started to internalize surface membrane and formed surface membrane invaginations closely attached to the outer rim of the cytoskeletal sphere which developed during the internal contraction. CF was internalized in these invaginations. CF was not observed within the system of dilated channels of stimulated platelets, however. Thrombin-stimulated gray platelets showed a markedly reduced number of dilated channels or none at all. This observation may be due to the fact “gray” platelets are deficient in α-granules. It is concluded that the dilated system of membranes in degranulated normal platelets originates from membranes of the α-granules which have performed compound exocytosis.