Inhibition Of Collagen-induced Platelet Aggregation Research Articles

An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis.

The saliva of Triatoma pallidipennis, a blood-sucking triatomine bug (Hemiptera, family Reduviidae, subfamily Triatominae) was found to contain a factor that specifically inhibits collagen-induced platelet aggregation. The 19-kDa protein was purified to homogeneity and named pallidipin. Collagen-mediated aggregation of platelets in plasma and of washed platelets was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors (ADP, thrombin, thromboxane A2 mimetic U46619, phorbol ester) was detected. Pallidipin had no effect on platelet adhesion to collagen but inhibited ATP release from platelets. It interacted reversibly with platelets and may share with collagen a common target on them. The protein exhibits a unique primary structure (predicted from cDNA clones) with no significant similarity to other previously described sequences. The protein produced in recombinant baby hamster kidney cells had antiaggregatory effects similar to those of native pallidipin. Availability of recombinant pallidipin will allow further investigation of the precise mechanism of action.

Antiplatelet constituents of formosan Rubia akane.

A known steroid, in addition to triterpenoids, anthraquinones, naphthalenes and a new anthraquinone glycoside, xanthopurpurin 3-O-beta-D-glucoside, were isolated from the roots of Rubia akane grown in Taiwan. Mollugin, a naphthohydroquinone, showed strong inhibition of arachidonic acid (AA)-induced and collagen-induced platelet aggregation. In contrast, 2-methyl-1,3,6-trihydroxyl-9,10-anthraquinone, xanthopurpurin 3-O-beta-D-glucoside, and xanthopurpurin showed mainly strong inhibition of collagen-induced platelet aggregation.

Inhibition of collagen-induced platelet aggregation and adhesion by a pseudocyanide derivative of avicine isolated from Zanthoxylum integrifoliolum MERR

Avicine pseudocyanide, a derivative of avicine isolated from Zanthoxylum integrifoliolum Men., inhibited collagen-induced platelet aggregation and release reaction in a concentration-dependent manner. Trimucytin is a collagen-like snake venom protein isolated from Trimeresurus mucrosquamatus. Avicine pseudocyanide also inhibited trimucytin (1 μg/mL)-induced platelet aggregation and release reaction concentration dependently. The ic 50 values of avicine pseudocyanide on collagen (10 μg/mL)-and trimucytin (1 μg/mL)-induced platelet aggregation were 47.3 ± 4.1 and 62.5 ± 5.6 μM, respectively. Avicine pseudocyanide at a concentration of 300 μM inhibited less than 30% of platelet aggregation induced by ADP (20 μM), AA (100 μM), U46619 (1 μM), PAF (2 ng/mL) and thrombin (0.1 U/mL). The concentration-response curve of collagen-induced platelet aggregation was shifted to the right by avicine pseudocyanide (20–100 μM) concentration dependently. The Schild plot showed that pA 2 and pA 10 values of avicine pseudocyanide were 4.8 and 4.3, respectively, with slope of −1.9. Avicine pseudocyanide also inhibited collagen (10 μg/mL)-induced aggregation of rabbit whole blood with an ic 50 of 145 ± 13 μM. Collagen-induced thromboxane B 2 formation was also inhibited by avicine pseudocyanide in a concentration-dependent manner with a maximal effect at 100 μM. However, arachidonic acid (AA)-induced thromboxane B 2 and prostaglandin D 2 formations were only partially suppressed by a high concentration of avicine pseudocyanide (300 μM). Avicine pseudocyanide (100 μM) inhibited the [ 3H]inositol monophosphate formation and the rise of intracellular Ca 2+ concentration caused by collagen but not those caused by AA, U46619, platelet-activating factor and thrombin. In the presence of prostaglandin E 1, Mg 2+-dependent platelet adhesion to collagen was inhibited by avicine pseudocyanide with an ic 50, of 278 ± 16 μM. These data indicate that avicine pseudocyanide is an inhibitor of collagen-induced platelet aggregation and platelet-collagen adhesion.

Cloning of the cDNA and expression of moubatin, an inhibitor of platelet aggregation.

Moubatin, a new type of specific inhibitor of collagen-induced platelet aggregation, has been isolated from the soft tick Ornithodoros moubata (Waxman, L., and Connolly, T. M. (1993) J. Biol. Chem. 268, 5445-5449). A polymerase chain reaction-generated hybridization probe, produced using primers based on moubatin protein sequence, identified phage containing the entire cDNA sequence of moubatin. Analysis of the predicted amino acid sequence yielded a mature protein of 156 amino acids with a putative prepeptide of 15 amino acids. Comparison of the sequence of moubatin to that of other proteins in the Swiss PROT data base revealed no significant homology. The cDNA sequence was cloned into the yeast expression vector pKH4 alpha 2, producing a biologically active protein which inhibited collagen-stimulated aggregation of washed human platelets with an IC50 of about 100 nM, which is similar to the potency of native tick moubatin. A concentration of recombinant moubatin that fully inhibited collagen-stimulated aggregation did not inhibit aggregation induced by a variety of other platelet agonists, again demonstrating comparable properties of the recombinant and native proteins. Moubatin did not inhibit platelet adhesion to collagen even at a concentration up to 16 times its IC50 for the inhibition of aggregation. This specificity for inhibiting collagen-stimulated aggregation and not adhesion to collagen indicates that moubatin is unique among the natural product inhibitors of collagen stimulation of platelets. Further examination of the mechanism of moubatin-mediated inhibition of collagen-stimulated aggregation revealed that 1-6 microM moubatin diminished the second phase of aggregation induced by ADP, inhibited aggregation in response to submaximal concentrations of the thromboxane A2 mimetic U46619, and competed for the binding of a thromboxane A2 receptor antagonist to platelet membranes. Therefore, at higher concentrations, moubatin may affect more than one aspect of platelet signal transduction including the thromboxane A2 receptor. The availability of recombinant moubatin will allow further investigation of its unique activities in vitro and in vivo.

S-nitroso-glutathione inhibits platelet activation in vitro and in vivo.

1. The effect of S-nitroso-glutathione (GSNO), a stable nitrosothiol, on platelet activation was examined in vitro and in vivo. 2. The adhesion of human platelets to fibrillar collagen and human endothelial cell monolayers was inhibited by GSNO. 3. GSNO caused a concentration-dependent inhibition of collagen-induced platelet aggregation in vitro and decreased ADP-induced aggregation in the conscious rat. 4. Inhibition of platelet aggregation in vitro correlated with the increase in intraplatelet cyclic GMP levels. 5. The release of NO from GSNO was enhanced by platelet lysate, native glutathione and ascorbate. 6. The results show that GSNO is a carrier of NO and therefore has pharmacological activity as an inhibitor of platelet activation.

5-[6-1 -(Cyclohexyl-1 H-tetrazol-5-YL)hexyl]-1,8-naphthyridin-2-(1H)-one, SC-44368, a Potent Anti-aggregatory Agent which Selectively Inhibits Platelet Cyclic AMP Phosphodiesterase

SC-44368 (5-[6-(1-cyclohexyl-1H-tetrazol-5-y)hexyl]-1,8-naphthyridin-2(1H)-one) is a potent and selective competitive inhibitor of platelet cyclic AMP-dependent phosphodiesterase (cAMP-PDE) (Ki: 1.65 μM). For the phosphodiesterase isoenzyms from human platelets SC-44368 shows a 26-fold selectivity (IC50 ratio) for the inhibition of the cAMP-PDE over the cyclic GMP-dependent phosphodiesterase (cGMP-PDE). By comparison, 3-isobutyl-1-methyl-xanthine (IBMX) inhibited the cAMP-PDE and cGMP-PDE from human platelets with approximately equal efficacy. Broad inhibitory activity was evident against human platelet aggregatory responses in vitro. IC50 values of 18.1 ± 5.3 μM (25 nM platelet activating factor, PAF), 17.3 ± 3.0 μM (1.0 μg/ml collagen) and 24.2 ± 10.3 μM (1μM ADP) were obtained against maximum increases in platelet-rich plasma (PRP) light transmission achieved by each agonist. SC-44368 potentiated the prostacyclin-induced increase of intra-platelet cAMP levels but did not potentiate the sodium nitroprusside-induced increase of intraplatelet cGMP levels. In an ex vivo model of platelet aggregation SC-44368 (3 mg/kg, i.v.) produced a potent inhibition of collagen-induced platelet aggregation. SC-44368 produced only weak hypotensive activity in the rat. Thus, SC-44368 is a novel cAMP-PDE inhibitor which possesses potent, broad spectrum anti-aggregatory properties.

Secretion of hementin and other antihaemostatic factors in the salivary gland complex of the giant amazon leech Haementeria ghilianii

Biological observations were made on the antihaemostatic activity in the saliva of the giant Amazon leech Haementeria ghilianii. Particular emphasis was placed on determining where the fibrino(geno)lytic enzyme hementin is produced and secreted in the salivary gland complex. Hungry third-fed Amazon leeches produce about 650 units of hementin, with by far the majority of the activity (75%) in the two posterior glands. The two anterior glands contain much less hementin (12%), and a small amount (12%) resides in the proboscis itself, presumably in the salivary gland ductules. In the anterior gland, hementin appears to be produced by only certain cells. The secretion of hementin is confined to the lumen of the proboscis. Secretions in the proboscis lumen are rich in antihaemostatic activity, as evidenced by fibrinogenolysis (hementin), prolongation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin clotting time (TCT) and Atroxin clotting time, and inhibition of collagen-induced platelet aggregation. In contrast, no anti-haemostatic, including hementin, activity was detectable at the tip. Suprisingly, the wound from the bite by the Amazon leech is not associated with prolonged bleeding. This is in marked contrast to the wound by the European medicinal leech Hirudo medicinalis which bleeds for an average of ten hours. The absence of bleeding is compatible with the interpretation, based on the above findings, that during feeding the Amazon leech does not inject antihaemostatic factors into the host, or at least not in the vicinity of the wound.

Tripyl-convergent total synthesis of a homochiral benzoindane-fused prostacyclin analog

Benzoindane-fused prostacyclin analog 14 was prepared from homochiral ammonium salt 5 in 7 steps and 35% overall yield. Key include addition of “soft” aryllithium reagent 6b to 5 to afford 7 ; and addition of homochiral acetylide 9 to the vinyl sulfone moiety of chloride 8 (with subsequent in situ cyclization) to afford 10 , thereby rapidly assembling the tricyclic framework of 14 . Compound 14 was a weak inhibitor of collagen-induced platelet aggregation having an IC 50 = 2.5μM.

Triply-convergent synthesis of two sets of homochiral cyclopent[a]indene carbacyclin analogs

Reaction of o -chloromethyl aryl cuprate 6 with optically active ammonium salt 7 regio- and stereospecifically affords vinyl sulfone 8 . Conjugate-addition of acetylenic anion 9 followed by in situ cyclization provides sulfone 10 which is refunctionalized to provide two sets of carbacyclin analogs, 15 - 17 and 25 - 27 . IC 50 values of these six compounds for inhibition of collagen-induced platelet aggregation were 0.18, > 100, 0.33, 0.33, > 100, and 2.2μM, respectively.

Triply-convergent syntheses of two homochiral arene-fused prostacyclin analogs related to U68,215

The syntheses of arene-fused prostacyclin analogs 2 and 3 are described. Rapid assembly of the tricyclic skeleton is achieved in a triply-convergent manner from oxazoline-bearing homocuprate 6b , homochiral ammonium salt 7 and homochiral propargyl acetylide 12 . Compound 2 was a potent inhibitor of collagen-induced platelet aggregation having an IC 50 = 2.9nM; while 3 exhibited an IC 50 = 52nM.

Total synthesis of a homochiral arene-fused prostacyclin analog

Reaction of aryl cuprate reagent 2b with homochiral allyl 3 ammonium salt 3 stereospecifically affords arene 4 . Conversion of this intermediate to chloride 6 followed by a conjugate-addition/cyclization protocol generates tricyclic sulfone 8 . Refunctionalization of this material provides homochiral prostacyclin analog 13 . Compound 13 was inactive as an inhibitor of collagen-induced platelet aggregation having an IC 50 > 10μM.

Syntheses and platelet aggregation inhibitory and antithrombotic properties of [2-[(.omega.-Aminoalkoxy)phenyl]benzenes

A series of [2-[(omega-aminoalkoxy)phenyl]ethyl]benzene derivatives were synthesized and evaluated for their ability to inhibit collagen-induced platelet aggregation in vitro and to protect experimental thrombosis in mice. The results showed that the compounds were in vitro inhibitors of collagen-induced platelet aggregation. Most of them were also effective in the mouse antithrombotic assay. The compounds were found to be potent antagonists to S2 serotonergic receptor, and good correlation (r = 0.85) between their S2 serotonergic receptor antagonism and their potency as platelet antiaggregatory drugs was observed. Among the compounds studied, mono[2-(dimethylamino)-1-[[2-[2-(3- methoxyphenyl)ethyl]phenoxy]methyl]ethyl] succinate hydrochloride (12b, MCI-9042) was selected for further pharmacological and toxicological evaluation.

Formula omitted] snake venom: Effects on platelet aggregation

Crude venom from Bothrops jararaca has procoagulant, platelet aggregating and phospholipase A2 (PLA2) activities. By chromatographic fractionation of the venom on Sephacryl S-200 it was possible to separate these three activities and to show that distinct protein components are involved. The procoagulant activity appears to involve the synergistic action of several components and was not further studied in the present work. The aggregating activity results from the action of two components: 1) a serino-proteinase PMSF-inhibitable similar to thrombocytin and 2) a PMSF-resistant, calcium- and plasma-dependent factor distinct from other previously described aggregating principles. Fractions possessing PLA2 activity were also able to inhibit platelet aggregation induced by collagen and accelerated the slow reversal of aggregation Induced by ADP. Both PLA2 activity and inhibition of collagen-induced platelet aggregation displayed by these fractions were abolished by reaction with p-bromophenacyl bromide and 2-mercaptoethanol. These results indicate that in B. Jararaca venom the PLA2 activity and the factor inhibiting platelet aggregation may be related to the same protein molecule.

Effect of labetalol on human platelet function.

1. The effects of the alpha-beta-adrenergic antagonist labetalol on the activation of human platelets by adrenaline and other aggregating stimuli have been investigated. 2. Labetalol inhibited platelet aggregation and secretion induced by collagen and the second phase of aggregation caused by ADP, platelet activating factor, adrenaline and ionophore A23187. Adrenaline-induced platelet activation was inhibited by the lowest labetalol concentration. The response to Na arachidonate was minimally affected and the arachidonate-induced thromboxane B2 generation was only partially prevented. 3. The pre-incubation with low concentration of ionophore A23187 overcame labetalol's inhibition of collagen-induced platelet aggregation. 4. Labetalol did not influence intraplatelet cyclic AMP levels. 5. The present investigation provided evidences that the modulation of human platelet function by labetalol could be related also to a decreased Ca2+ availability.

Human blood platelets possess specific binding sites for C1q.

Although platelet interactions with C1q are implied by the inhibitory effect of C1q on collagen-induced platelet aggregation, specific receptors have not as yet been identified. To address the question of platelet receptors for free C1q, direct radioligand binding studies were performed by using human blood platelets and purified, 125I-labeled C1q, and a monoclonal antibody (II1/D1) (IgM, lambda) directed against C1q receptors on peripheral blood leukocytes. Washed platelets bound both purified 125I-labeled C1q and II1/D1 in a specific and saturable manner under physiologic ionic strength conditions. At equilibrium, approximately 4000 molecules of C1q bound per platelet with an apparent dissociation constant of 3.5 X 10(-7) M. Maximum C1q binding was achieved in 5 min and correlated well with inhibition of collagen-induced platelet aggregation. Equilibrium binding of 125I-labeled II1/D1 to washed platelets required an incubation period of 15 to 30 min and II1/D1 concentrations approaching 50 micrograms/ml. Approximately 2000 molecules of II1/D1 bound per platelet, with an apparent dissociation constant of 2.8 X 10(-8) M. II1/D1 binding could be inhibited by the collagenous tail of C1q (c-C1q), suggesting that platelet receptors for these ligands are either the same or in close proximity. The data demonstrate that human blood platelets possess specific and saturable binding sites for free C1q that may function as collagen receptors, and may antigenically resemble C1q receptors on peripheral blood leukocytes.

Free Platelet Count and Size Distribution During C1q Inhibition of Collagen-Induced Platelet Aggregation

Electronic free platelet counting was more sensitive than turbidimetry to detect collagen-induced platelet activation in human platelet-rich plasma. Purified human C1q exhibited a greater inhibitory effect on collagen-induced platelet aggregation in turbidimetry than free platelet counting. Because the change from small to large platelet aggregates is responsible for the continuing increase in light transmission, C1q was likely more capable of blocking the formation of large platelet aggregates than the formation of small aggregates from single platelets. The rate of change by collagen in light transmission and free platelet count was reduced in the presence of C1q but the timing of the peak response remained the same. Electronic platelet sizing revealed that the volume of single platelets transiently increased during the turbidimetric "lag phase". The mean, mode and median volume of the remaining free platelets then decreased, suggesting a selective loss of large, functionally more active platelets and/or platelet degranulation. C1q had no effect on the volume increment during the "lag phase", but reduced the subsequent fall in the volume of free platelets.

ChemInform Abstract: SYNTHESIS AND PLATELET AGGREGATION INHIBITORY ACTIVITY OF 4,5‐BIS(SUBSTITUTED)‐1,2,3‐THIADIAZOLES

AbstractDie durch Cyclisierung der Hydrazone (III) zugänglichen Thiadiazo‐ Ie (IV) werden mit der antithrombotisch wirkenden Verbindung (V) verglichen.

Synthesis and platelet aggregation inhibitory activity of 4,5-bis(substituted)-1,2,3-thiadiazoles.

Routine screening of compounds for inhibition of collagen-induced platelet aggregation in vitro revealed 4,5-bis-(4-methoxyphenyl)-1,2,3-thiadiazole (2) was active and it represents the first example of a 1,2,3-thiadiazole with possible antithrombotic activity. In order to develop a structure-activity relationship for this heterocycle, a number of new 4(5)-mono- and -disubstituted 1,2,3-thiadiazoles were synthesized. These were tested in our screen and a number of additional active compounds were found. The most active compounds (2, 5a, 5b, and 6c) were those in which the heterocycle was substituted with benzene rings possessing para electron-donating groups.

Interaction of specific platelet membrane proteins with collagen: evidence from chemical cross-linking.

Two recently developed membrane-impermeant cross-linkers, 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) and bis(sulfosuccinimidyl) suberate (BS3), have been used to examine the interaction of human platelets with collagen. Reaction of human platelets with either of the two cross-linking reagents at micromolar concentrations completely inhibited platelet aggregation in response to collagen but not in response to thrombin. Platelet adhesion to collagen was, however, not affected by these reagents. Inhibition of collagen-induced platelet aggregation by DTSSP or BS3 appears to be due to cross-linking and not simply to the chemical modification of membrane proteins, since the homologous monofunctional reagent sulfosuccinimidyl propionate had no effect on platelet aggregation. Inhibition of platelet aggregation by BS3 was accompanied by a decrease in the intensity of glycoprotein bands IIb, IIIa, and IV when analyzed on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels. In order to determine if collagen is directly interacting with a specific platelet membrane glycoprotein, 3H-labeled platelets were allowed to adhere to collagen and then cross-linked with various concentrations of DTSSP. Proteins which remain associated with collagen after lysis and washing were analyzed on NaDodSO4 gels. At concentrations of 16-50 microM DTSSP, glycoproteins IIb and IIIa appeared to be specifically cross-linked to collagen. These results suggest that the glycoprotein IIb-IIIa complex, which has previously been implicated as the fibrinogen receptor in activated platelets, may also be directly involved in collagen-induced platelet aggregation.

Effects of Acetylsalicylic Acid on Human Platelet Function In Vivo at Salicylate Steady State

The results of clinical trials concerning the use of acetylsalicylic acid (ASA) as antithrombotic drug are contradictory. Inhibition by ASA of platelet prostaglandin synthesis and aggregation is prevented by its metabolite salicylic acid (SA) in animals and in human platelets in vitro. It was suggested that ASA might produce its own inhibitor, thereby diminishing its efficiency in thromboembolic disease. In four healthy male subjects there was no difference in inhibition of collagen-induced platelet aggregation after the administration of 500 mg ASA alone or at salicylate steady state (3 g SA daily). But the inhibition of tissue-extract-induced platelet shape change was diminished and shortened by pretreatment with SA. We conclude that SA does not inhibit the effects of ASA on human platelet aggregation in vivo in therapeutic dose ranges. The clinical importance of the SA/ASA-interaction on tissue-extract-induced platelet shape change remains to be clarified.