Inhibition Of Thrombin-induced Platelet Aggregation Research Articles

Cultured astrocytoma cells inhibit platelet aggregation by releasing a nitric oxide-like factor

Cultured astrocytoma cells were tested for their ability to generate a nitric-oxide like factor using platelet aggregation as a bioassay. Incubation of astrocytoma cells with human washed platelets resulted in an inhibition of thrombin-induced platelet aggregation which was proportional to the number of astrocytoma cells added. The inhibition was potentiated by superoxide dismutase (SOD) and prevented by oxyhaemoglobin (oxyHb). The inhibitory activity of astrocytoma cells was also prevented by the NO biosynthesis inhibitor N G-monomethyl-L-arginine (MeArg), an effect reversed by co-incubation with L-arginine (L-Arg) but not D-arginine (D-Arg). These results demonstrate that astrocytoma cells release, independent of added agonist, a factor with the same pharmacological profile as NO, which is likely to be derived from L-arginine.

Immediate release of a nitric oxide-like factor from bovine aortic endothelial cells by Escherichia coli lipopolysaccharide.

Incubation of human washed platelets with bovine aortic endothelial cells (ECs) treated with indomethacin resulted in an inhibition of thrombin-induced platelet aggregation that was dependent on the number of ECs added. Preincubation of ECs with Escherichia coli lipopolysaccharide (LPS; 0.5-2.0 micrograms/ml) for 1 min significantly enhanced their inhibitory activity. This effect was potentiated by superoxide dismutase (60 units/ml) and reversed by oxyhemoglobin (5-10 microM), indicating that the inhibition was due to the release of endothelium-derived relaxing factor (nitric oxide). When the ECs were pretreated with NG-monomethyl-L-arginine (30-300 microM) before LPS, the antiaggregatory activity was strongly reduced. The reduction of activity by NG-monomethyl-L-arginine was reversed by L-arginine (100 microM) but not by D-arginine (100 microM). Under similar conditions, LPS also enhanced the antiaggregatory activity of ECs grown on beads. The immediate enhancement by LPS of the release of endothelium-derived relaxing factor from endothelial cells may contribute to the rapid fall in blood pressure associated with endotoxin shock in vivo.

Platelet activating factor and sheep platelets: A sensitive new bioassay

The in vitro response of sheep platelets to platelet activating factor (PAF) was investigated. Sheep platelet-rich plasma aggregated in response to PAF with an EC 50 of 10 nM. Platelets isolated via arabinogalactan density gradient centrifugation displayed an EC 50 of 50 nM with a threshold response at 0.1 pM. PAF-induced release of 14C-serotonin from isolated sheep platelets was comparable with an EC 50 of 50 pM and threshold release at 10 fM. PAF-induced aggregation was specific in that it could be blocked by the competitive receptor antagonists Alprazolam (Upjohn, IC 50 = 40 μM), L-652, 731 (MSD, IC 50 = 6 μM), and WEB 2086 (Boehringer Ingelheim, IC 50 = 0.8 μM). At micromolar concentrations, WEB 2086 did not inhibit ADP- or thrombin-induced aggregation nor thrombin-induced serotonin release. However, at higher concentrations of WEB 2086 some inhibition of thrombin-induced platelet aggregation and release was observed. Subsequent experiments demonstrated that high concentrations of WEB 2086 can inhibit thrombin-induced clotting (K i = 866 μM) and cleavage of the chromogenic substrate Spectrozyme-TH (K i = 842 μM). In summary, the response of isolated sheep platelets to PAF was specifically inhibitable and was 10 to 100 times more sensitive than washed rabbit platelets, the most popular bioassay currently in use.

Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxide-like factor.

Incubation of neutrophils or mononuclear cells with washed platelets (all prepared from human venous blood) resulted in an inhibition of thrombin-induced platelet aggregation that was dependent on the number of nucleated cells added. The inhibition was potentiated by superoxide dismutase and reversed by oxyhemoglobin. In the case of neutrophils the inhibition was associated with an increase in cGMP, whereas with mononuclear cells both cAMP and cGMP were increased. The inhibitory activity of neutrophils or mononuclear cells was prevented by their preincubation with NG-monomethyl-L-arginine methyl ester. L-Arginine reversed the action of NG-monomethyl-L-arginine methyl ester, whereas D-arginine was ineffective. Preincubation of the cells with catalase or mannitol did not prevent their inhibitory action on platelet aggregation. The inhibition of platelet aggregation was not due to platelet damage or to uptake of thrombin by neutrophils or mononuclear cells. It was overcome by increasing the concentration of thrombin and was absent in cell-free supernatants obtained from a suspension of neutrophils or mononuclear cells or from mixtures of platelets with neutrophils or platelets with mononuclear cells. These data provide evidence for the release of a nitric oxide-like factor from human neutrophils and mononuclear cells. In addition, evidence is provided that, as in stimulated murine macrophages and endothelial cells, the precursor of this factor is L-arginine.

Inhibition of thrombin-induced platelet aggregation by high molecular weight kininogen.

5′-p-Fluorosulfonylbenzoyl adenosine (FSBA) is a potent inhibitor of platelet shape change, aggregation and fibrinogen binding by a mechanism which has been linked to the covalent modification of a single surface membrane polypeptide, aggregin (Mr = 100 kD)1,2. We recently demonstrated that cleavage of aggregin in [3H]FSBA-labeled platelets during platelet aggregation induced by thrombin indirectly involves the intracellularly activated calcium-dependent cysteine protease, calpain3 and is mediated by the high affinity thrombin receptor4. Schmaier et al.5 showed that high molecular weight kininogen (HK) is the most potent plasma inhibitor of platelet calpain. We now report that inhibition of thrombin-induced cleavage of aggregin by HK during platelet aggregation involves specific inactivation of platelet calpain.

Inhibition of thrombin-induced platelet aggregation by myristic acid

Myristate inhibited thrombin-induced aggregation and production of diacylglycerols of rabbit platelets suspended in a modified Tyrode solution. The incorporation of inositol into platelet phospholipids in both steady and activated states was also inhibited by myristate. When platelets were incubated with 32p-inorganic phosphate in the presence of myristate, incorporation of the radioactivity into platelet phosphatidylinositols (PI) was distinctly inhibited, although that into phosphatidic acid (PA) was inhibited relatively little. These results suggest that thrombin-induced breakdown of inositol phospholipids and the metabolic pathway of PA to PI are inhibited by myristate. The saturated fatty acids which have lower and higher moleculer weight than myristate revealed a little or no inhibitory effects on the aggregation and on these metabolic changes of platelet lipids, suggesting the specificity of chain-length in the inhibitory action. It is conceivable that the inhibition of thrombin-induced aggregation by myristate is associated with the inhibition of PI-turnover in the platelets treated with the fatty acid.

In Vivo Effects of a Low Molecular Weight Heparin Fragment on Platelet Aggregation and Platelet Dependent Hemostasis in Dogs

Plasma defibrinogenated dogs were used to study the influence of conventional heparin and a low molecular weight heparin fragment (Fragmin, mean MW 5,000 d) on platelet dependent hemostasis. The heparins were given intravenously in gravimetrically equal doses. The bleeding from standardized skin flap wounds and platelet aggregation (ADP and thrombin) was studied. In comparison, higher doses of the fragment than of heparin were required to increase the bleeding. ADP-induced aggregation in defibrinogenated platelet rich plasma (after addition of normal dog plasma) was potentiated by both heparins. After injection of heparin or the fragment, ADP induced platelet aggregation without prior addition of normal plasma to the test-tube. In conclusion the heparin fragment affected bleeding to a less extent than conventional heparin. One explanation might be a weaker inhibition of thrombin-induced platelet aggregation.

Impaired Platelet Aggregation and Thromboxane Generation in Essential Fatty Acid—Deficient Rats

ADP-induced platelet aggregation and thrombin-induced thromboxane B2 generation in diluted whole blood from rats fed a fat-free diet supplemented with 10% (by weight) hydrogenated coconut oil [essential fatty acid (EFA) deficient] were significantly lower than that in animals fed 10% safflower oil [(SFO) rich in linoleic acid] or 10% marine oil (rich in eicosapentaenoic acid and docosahexaenoic acid). Plasma fibrinogen levels were significantly lower and liver function was impaired in the EFA-deficient group compared with the other two groups. Platelet responsiveness to ADP was restored when plasma from the EFA-deficient rats was replaced by plasma obtained from rats fed a nonpurified diet. ADP responsiveness and thrombin-stimulated thromboxane B2 production in diluted whole blood were also restored after 2 wk of injections of 100 mg ethyl linoleate every 48 h, and partially restored by injections of 100 mg ethyl α-linolenate. When ADP-induced platelet aggregation was examined in washed platelets, the impaired ADP aggregation (found when platelets of the EFA-deficient rats were suspended in their own plasma) was not observed at either high (9.5 µM) or low (1.0 µM) ADP concentrations. Although thrombin-stimulated thromboxane B2 production in washed platelets in the EFA-deficient rats was lower than that in the SFO-fed rats, the magnitude of aggregation was not different. In addition, inhibition of thrombin-induced platelet aggregation by apyrase (0.06 u/mL) was identical in the two groups. These results suggest that impaired platelet aggregation in EFA deficiency is related more to plasma factors than to inherent platelet properties and that restoration of normal liver function is associated with normal platelet function. Fibrinogen may play a role. The combined effects of reduced arachidonic acid composition in platelet phospholipids and impaired thrombin-ADP synergism in platelet aggregation seem to be responsible for the decreased thromboxane B2 production, but this may not be of major importance in impairing platelet function in EFA deficiency.

Inhibition of thrombin-induced platelet aggregation by uteroglobin

Uteroglobin, a steroid-dependent, small molecular weight (15 K) protein in the rabbit, inhibited thrombin-induced aggregation of both rabbit and human gel-filtered platelets (GFP). GFP aggregation by arachidonic acid was not affected by uteroglobin. There were no effects of uteroglobin on thrombin-induced clotting of plasma or purified fibrinogen, or inhibition of thrombin by antithrombin III. Additionally, preliminary results suggest that uteroglobin does not interfere with binding of thrombin to platelets. We suggest that inhibition of platelet aggregation by uteroglobin may function in preventing thrombosis and ensuring free flow of blood through the microvasculature of the uterus and the placenta and may induce some of the antimotility effects of progesterone on the uterus.

Protease and cyclooxygenase inhibitors synergistically prevent activation of human platelets.

Thrombin induces platelet aggregation and formation of a fibrin clot in platelet-rich plasma; leupeptin, a protease inhibitor, partially inhibits platelet aggregation, but it does not inhibit fibrin clot formation. Indomethacin does not inhibit either thrombin-induced platelet aggregation or fibrin clot formation. However, when the two drugs are given together, a synergistic inhibition of thrombin-induced platelet aggregation occurs, while fibrin clot formation remains unaffected. Thrombin-induced stimulation of the release of serotonin in washed human platelets is also synergistically inhibited by the combined actions of leupeptin and indomethacin. Thrombin and collagen, added simultaneously, induce full platelet aggregation and release of serotonin. Neither leupeptin nor indomethacin inhibits platelet responses elicited by both agonists; however, when leupeptin and indomethacin are given together, a synergistic inhibition of thrombin- and collagen-induced response is observed. These findings might be relevant in prophylaxis and treatment of thromboembolic disease.

Inhibition of thrombin-induced platelet aggregation and serotonin release by antithrombin III and heparin cofactor II in the presence of standard heparin, dermatan sulfate and pentosan polysulfate

Inhibition of thrombin-induced platelet aggregation and serotonin release by antithrombin III and heparin cofactor II in the presence of standard heparin, dermatan sulfate and pentosan polysulfate

Effects of antiplatelet agents on pulmonary metastases.

The role of platelets in cancer metastasis was studied by investigating the effects of the antiplatelet agents ticlopidine, diltiazem, dipyridamole and trapidil on artificial and spontaneous pulmonary metastases in mice. These agents were tested at their optimal inhibitory doses on adenosine diphosphate-induced platelet aggregation; namely, 100 mg/kg for ticlopidine, 2 mg/kg for diltiazem, 180 mg/kg for trapidil and 60 mg/kg for dipyridamole. At these doses, trapidil caused moderate inhibition of thrombin-induced platelet aggregation in mice, but the other agents had only slight effects. Artificial pulmonary metastasis was produced by inoculation of Lewis lung carcinoma (LLC) or B16 melanoma (B16) cells into C57BL/6 mice. For induction of spontaneous pulmonary metastases, these tumor cells were implanted subcutaneously into the footpads of mice. The resulting primary tumors of LLC and B16 were removed 9-10 and 17 days later, respectively. Artificial pulmonary metastases were inhibited significantly by all the antiplatelet agents tested. Spontaneous pulmonary metastases were markedly reduced only when these agents were given after removal of the primary tumor. The role of platelets is discussed with respect to thrombus formation in the lodgement of tumor cells and the participation of platelet-derived growth factor in the growth of metastatic foci.

Inhibition of Thrombin-Induced Platelet Aggregation by a Derivative of Wheat Germ Agglutinin. Evidence for a Physiologic Receptor of Thrombin in Human Platelets

We heve prepered a nonagglutinating derivative of wheat germ agglutinin by cyanogen bromide treatment of the lectin and used it as a probe to study the thrombin-platelet interaction. The lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by collagen, ristocetin, adenosine diphosphate, wheat germ agglutinin, or trypsin was not significantly affected. Under similar conditions, the secretion of serotonin by thrombin was blocked by the derivative. The inhibitory action of the derivative on thrombin-induced platelet aggregation could be overcome by increasing the thrombin concentration. A Schild plot of these data yielded a slope of 1.0 and an apparent dissociation constant of 1.0 µM. Thus, the inhibition of thrombin-induced platelet aggregation by the derivative fits a model of competitive inhibition. Control experiments showed that the lectin derivative acted on platelets and not on thrombin. However, the binding of [125I]thrombin to platelets was not affected. Isolated platelet membranes were solubilized and passed through a column of the lectin derivative coupled to Sepharose. After extensive washing, the material bound to the column was eluted with N-acetyl-d-glucosamine. This membrane isolate inhibited platelet aggregation by thrombin while aggregation induced by adenosine diphosphate, trypsin or ristocetin was not significantly affected. It also blocked thrombin-induced serotonin secretion from platelets. Gel electrophoresis followed by autoradiography of the membrane isolate revealed a prominent glycoprotein of apparent molecular weight of 74,000 that contained inhibitory properties. The electrophoretic mobility or the intensity of this band was not significantly affected following incubation of the glycoprotein isolate with physiologic concentrations of thrombin. The glycoprotein also retained its inhibitory activity on thrombin-induced platelet aggregation following incubation with 20 µg/ml of trypsin or chymotrypsin for 10 min. These results suggest that (A) the thrombin receptor in human platelets is different from the ristocetin or ristocetin-von Willebrand factor receptor and (B) the 74,000 dalton glycoprotein may be a physiologic receptor of thrombin in human platelets.

Inhibition of thrombin-induced platelet aggregation by a derivative of wheat germ agglutinin. Evidence for a physiologic receptor of thrombin in human platelets

Inhibition of thrombin-induced platelet aggregation by a derivative of wheat germ agglutinin. Evidence for a physiologic receptor of thrombin in human platelets

Mechanism of inhibition of thrombin-induced platelet aggregation by pyridoxal phosphate

Thrombin and ADP-induced platelet aggregation are reversibly inhibited by pyridoxal phosphate. Sodium borohydride converts Schiff bases formed between pyridoxal phosphate and amino groups to covalent bonds. When platelets treated with sodium borohydride and pyridoxal phosphate are resuspended in fresh platelet-poor plasma, they recover their response to thrombin, but not to ADP. Thus Schiff base formation between pyridoxal phosphate and platelet surface amino groups does not block thrombin aggregation. The loss of thrombin potency as an aggregating agent is due to interaction between pyridoxal phosphate and thrombin. This is evidenced by spectrophometric determination of adduct formation and loss of hydrolytic action on p-tosyl-L-arginine methyl ester.

Effects of Prostaglandins, Derivatives of Cyclic 3':5'-AMP, Theophylline, Cholinergic Agents and Colchicine on Clot Retraction in Dilute Platelet-Rich Plasma and Gel-Separated Platelet Test Systems

In dilute suspensions of platelet-rich plasma (PRP) or gel-separated platelets (GSP), dibutyryl-cAMP (DBcAMP) and monobutyryl-cAMP inhibited platelet-mediated fibrin clot retraction in concentrations of 2--3 X 10(-6) M, with complete inhibition at 1--3 X 10(-4) M. Prostaglandin E1 (PGE1), which inhibited fibrin clot retraction in concentrations greater than 1.5--3 X 10(-8) M, was a more effective inhibitor than either PGE2 or PGF2 alpha. In the presence of theophylline (10-4 M), concentrations of DBcAMP, PGE1, PGE2 and PGF2 alpha necessary to inhibit fibrin clot retraction were reduced 50-fold for DBcAMP and 2.5 to 20-fold for the prostaglandins. In dilute PRP or GSP, inhibition of fibrin clot retraction does not result from inhibition of thrombin-induced platelet aggregation. Thus, compounds which increase platelet cAMP levels result in the inhibition of platelet-mediated fibrin clot retraction, and this inhibitory effect may be mediated, at least in part, through suppression of platelet contractility. Cyclic GMP, dibutyryl-cGMP and carbamylcholine-Cl (which stimulate guanylate cyclase) did not influence fibrin clot retraction, and did not prevent inhibition of fibrin clot retraction by DBcAMP and PGE1. Colchicine, in concentrations known to disrupt platelet microtubules (2.5 X 10(-6) M to 2.5 X 10(-3) M), had little inhibitory effect on either fibrin clot retraction or platelet (3H)-serotonin release.

Plasmin Inhibition of Thrombin-induced Platelet Aggregation

The effects of plasmin treatment upon washed human platelets were studied in an attempt to elucidate the mechanisms underlying thrombin-induced platelet aggregation. At calcium concentrations of 10-20 muM, PLASMIN (0.2 CTA U/ml) inhibited thrombin-induced aggregation almost completely, but did not diminish the thrombin-induced release of adenine nucleotides, 5-hydroxytryptamine, or calcium. Increasing the calcium concentration partially antagonized plasmin's inhibition of aggregation. Studies utilizing calcium chelators and the Kunitz soybean trypsin inhibitor (SBTI) as a plasmin inhibitor indicated that in order to achieve maximal block of aggregation, plasmin must act upon a substrate made fully available only after an initial thrombin-platelet interaction has taken place. Moreover, the time course of this inhibition parallels the time course of the thrombin-induced release reaction. Plasmin inhibition of aggregation could not be mimicked by exposing the platelets to proteolytic digests of fibrinogen at concentrations as high as 17% total platelet protein. Nor could inhibitory activity be recovered from supernatants of plasmin-treated platelets, upon centrifugation and treatment with SBTI. With the use of a "cold initiation" technique, the release by thrombin of 46.7 plus or minus 6.7 (mean plus or minus SEM) mu-g of fibrinogen immunological equivalents per mg platelet protein could be demonstrated. Platelets in which thrombin-induced aggregation was abolished by plasmin treatment (and the plasmin subsequently inactivated by STBI) aggregated normally upon addition of as little as 10 mu-g human plasma fibrinogen per mg platelet protein. It is concluded that plasmin inhibition of aggregation most likely results from its attack upon a protein that is released or becomes fully available subsequent to interaction of thrombin with a platelet receptor mediating release. The results of this study are consistent with a cofactor role for fibrinogen in the aggregation of human platelets by thrombin.

Inhibition of ADP-induced Platelet Aggregation by Substituted Amino-acids

AGGREGATION of platelets in the presence of adenosine diphosphate (ADP) appears to be important in haemostasis and in the genesis of platelet thrombosis. There is evidence that this reaction is common to platelet clumping induced by a variety of stimuli, including collagen, adrenaline, long-chain fatty acids, and perhaps weak solutions of thrombin. Although this phenomenon is under investigation in many laboratories, the mechanism by which ADP causes platelet aggregation is largely unknown. Certain inhibitors of platelet clumping have been described (for example, inhibition of thrombin-induced platelet aggregation by heparin1 or of ADP by adenosine2), and investigation of the inhibitory reaction has afforded some insight into the underlying process.