Anticoagulant Activities of Indobufen, an Antiplatelet Drug.

Indobufen is a new generation of anti-platelet aggregation drug, but studies were not sufficient on its anticoagulant effects. In the present study, the anticoagulant activity of indobufen was determined by monitoring the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) in rabbit plasma. We evaluated the anticoagulant mechanisms on the content of the platelet factor 3,4 (PF3,4), and the coagulation factor 1, 2, 5, 8, 10 (FI, II, V, VIII, X) in rabbits, as well as the in vivo bleeding time and clotting time in mice. The pharmacodynamic differences between indobufen and warfarin sodium, rivaroxaban, and dabigatran were further studied on thrombus formation and the content of FII and FX in rats. Animal experiments showed that intragastric-administrated indobufen can significantly reduce the APTT, PT, TT, PF3, FI, II, V, VIII, and X plasma contents. Its inhibitory effect on plasma FII was better than thrombin inhibitor dabigatran with effect on FX better than FXa inhibitor rivaroxaban. These results suggest that indobufen has some anticoagulant effects as strong as some conventional anticoagulants. The mechanism may be related to both exogenous and endogenous coagulation system.

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo.

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles.

Platelet activation in Alzheimer disease.

BACKGROUND: In light of recent reports of diminished platelet serotonin concentration and increased plasma serotonin levels in patients with Alzheimer disease (AD), we hypothesized that a state of heightened platelet activation might be present in AD. OBJECTIVE: To compare baseline activation of unstimulated platelets in patients with AD with that in control subjects. PATIENTS AND METHODS: Flow cytometry was used to measure platelet activation in 91 patients with probable AD and 40 age-matched control subjects. Groups were compared for percentage of circulating platelet aggregates, expression of CD62p, formation of leukocyte-platelet complexes, and presence of circulating platelet microparticles, controlling for effects of demographic, clinical, physiological, and logistical factors. RESULTS: Multiple analysis of covariance on ranked data revealed a 39.5% increase in percentage of platelet aggregates (P=.0001), a 59.3% increase in expression of CD62p (P=.001), and a 53.3% increase in leukocyte-platelet complexes (P=.0001) in the group with AD but no differences in the number of platelet microparticles, overall platelet count, plasma fibrinogen level, or plasma platelet factor 3. Activation was weakly correlated with sex, but was independent of age, severity of disease, duration of disease, depression, agitation, and family history of dementia. CONCLUSIONS: Platelets of patients with AD exhibit greater unstimulated activation than those of controls. Potential causes of such activation include possible stimulation of platelets by damaged cerebral endothelial cells or platelet activation induced by membrane abnormalities previously reported to be present in platelets of patients with AD. In light of recent evidence that platelets are the principal source of both amyloid precursor protein and beta-amyloid peptide in human blood, it is possible that AD platelet activation may reflect or even contribute to the pathogenesis of the disease.

Thrombocytopenia, macrothrombocytopathia, nephritis and deafness.

The association of thrombocytopenia, macrothrombocytopathia, nephritis and deafness is rare. Reported here is a new case of this triple association. The clinical course, the nephropathologic findings and the bilateral neurologic hearing loss were similar to those already reported, with a slowly progressive impairment of renal function accompanied by a persistent proteinuria. The platelet diameters were increased. These macroplatelets contained granules of normal structure but with an irregular distribution in the cytoplasm. In other areas the cytoplasm was rich in surface connected system. The survival of these platelets and their contraction were normal. Their aggregation and excretion in response to collagen, adenosine diphosphate and thrombin, and the values of platelet factor 3 activity were all decreased. The degranulation defect, also present, was observed in the absence of a decrease in intracellular cyclic adenosine 5'-monophosphate (AMP) suggesting a relationship between these two findings.

Substituted alpah-methylbenzyl and tricyclic arylalkyl lactamimides as inhibitors of blood platelet aggregation.

N-[1-(p-Phenoxyphenyl)ethyl]hexahydro-2H-azepin-2-imine hydrochloride (10) and N-[1-(2-dibenzothienyl)-ethyl]hexahydro-2H-azepin-2-imine hydrochloride (22) were found to inhibit in vitro aggregation of human blood platelets induced by ADP with minimal release of procoagulant platelet factor 3. The compounds were selected from a series of substituted alpha-methylbenzyl and tricyclic arylalkyl lactamimides that were free of hypoglycemic and diuretic effects. Compounds 10 and 22, as well as N-[1-(1-naphthyl)ethyl]hexahydro-2H-azepin-2-imine hydrochloride (I) and N-(2,2-diphenylpentyl)hexahydro-2H-azepin-2-imine hydrochloride (II), were evaluated for effects on ADP-induced platelet aggregation after repeated oral administration to guinea pigs. Compound II (RMI 12,366A) showed in vivo activity in this system 2 h after the last of four daily doses of 100 mg/kg po.

(2-Piperidine)- and (2-pyrrolidine)ethanones and -ethanols as inhibitors of blood platelet aggregation.

(E)-4-[4-(Methylthio)phenyl]-1-(2-piperidinyl)-3-buten-2-one hydrochloride (44, RMI 14 133A) was found to inhibit ADP-induced aggregation of blood platelets. It was selected from a large series of (2-piperidinyl)- and (2-pyrrolidinyl)ethanones synthesized by a modified Schopf reaction from enolate magnesium salts of beta-keto acids and 2,3,4,5-tetrahydropyridine trimer or 3,4-dihydro-2H-pyrrole trimer, respectively. Evaluation of the compounds was carried out in vitro on human blood platelets. Structure-activity relationships are discussed. 44 also inhibited platelet aggregation ex vivo in guinea pigs. Subacute toxicity evaluation in dogs and guinea pigs showed it to have an unfavorable therapeutic ratio. 1-[4'-Chloro(1,1'-biphenyl)-4-yl-a1-2-(2-piperdinyl)ethanone hydrochloride (18, RMI 12436A) was found to lower serum cholesterol levles in rats with concurrent accumulation of (3beta)-cholesta-5,7-dien-3-ol, suggesting inhibition of 7-dehydrocholesterol delta7-reductase.

Differing susceptibilities of platelets from normal individuals and patients with von Willebrand's disease to attack by quinine- and quinidine-dependent antiplatelet antibodies.

Reactivity of quinine- and quinidine-dependent antiplatelet antibodies has been compared in platelet-rich-plasma (PRP) from normal donors and from patients with von Willebrand's disease (vWd). One quinine-dependent antibody (Q.Ab) caused platelet aggregation and [14C] serotonin release with only 7 of 12 normal donors, while another Q.Ab and a quinidine-dependent antibody (Qd.Ab) caused aggregation and release with all 12. Drug-dependent IgG binding and PF 3 availability induced by the antibodies were, however, comparable in all donors. Differences in responsiveness were associated with platelets and not plasma. vWd platelets showed normal drug-dependent IgG binding, but decreased aggregation and serotonin release to most drug-dependent antibodies. Responsiveness was not restored by purified vWf:Ag, but, in one case, was corrected by normal plasma or cryoprecipitate. Drug-dependent binding of the Q.Ab which caused variable responsiveness in normals was to the same platelet antigens (GPIb and GPIIIa) in both normal and vWd platelets and did not require plasma components. Reduced PF 3 availability was seen with some antibodies in some vWd patients. Plasma from two of these patients inhibited aggregation of normal platelets to Q.Ab and one of these inhibited aggregation to ADP. Antiplatelet antibodies were detected in these two plasmas by ELISA. Thus some Q.Ab produce different responses with platelets from different donors. In vWd, reduced responsiveness to Q.Ab and Qd.Ab may result from production of inhibitory antiplatelet antibodies.

Lipids as triggering factors in thrombosis.

An association has been established between acute and more persistent changes in lipid metabolism as reflected in plasma lipids, and platelet lipid metabolism. Platelet function is affected, particularly the activity and availability of platelet factor 3, however, also other changes making the platelets more sensitive to aggregating substances without interfering with the lipid part of platelet factor 3, have been documented. Experimental studies have demonstrated an increased tendency to thrombosis in animals given a diet with a high fat content with a high ratio of saturated to polyunsaturated fatty acids. Studies in man have mainly established a connection between dietary fats, plasma lipid abnormalities and frequency of coronary heart disease and clinical studies more directly relating thrombosis to lipid metabolism is highly warranted. Many open questions remain to be answered. Probably most relevant would be to understand how the antithrombotic mechanisms in the body are affected by changes in lipid metabolism. Even if thrombotic lesions are very common events in the western world our knowledge based on laboratory and experimental studies should indicate a much higher incidence, solely based on interactions between lipids and platelets in subjects exposed to our dietary habits and our way of life.

In vitro and in vivo effects of adrenaline and phentolamine on platelet function.

In vitro and in vivo effects of adrenaline (ADR) on platelet aggregation, on platelet factor 3 (PF3) availability and on platelet factor 4 (PF4) release were studied in man. Inhibitory action of an alpha-blocker, phentolamine (PHEN) was investigated in the same conditions. The threshold concentration (TC) of ADR inducing the typical two-phase response in aggregation tests when added to platelet-rich plasma (PRP) varied in different pools of plasma, but always induced an evident PF4 release and increased PF3 availability. A further increase in both parameters was obtained with higher concentrations but without any significant dose/response correlation. Adding PHEN alone to PRP did not induce platelet aggregation or modify PF4 release induced by stirring, but it reduced PF3 availability. On the other hand, PHEN prevented the effects of ADR in different platelet tests, at appropriate concentrations. Intravenous infusion of ADR lowered the TC, and increased PF3 availability and PF4 release. In vivo administration of PHEN, in contrast, increased TC and reduced PF3 availability, while PF4 remained unchanged.

Generation of a PGI2-like activity by deendothelialized rat aorta.

We have tested a platelet aggregation inhibitor in the incubation fluid of deendothelialized fragments of the rat aorta and compared it with that of "intact" fragments. Some of the properties of the aortic inhibitor, and its effects on platelet adhesion to collagen fibrils, on platelet factor-3 (PF-3) availability, and on the activated partial thromboplastin time (APTT) and thrombin time (TT) were also evaluated in comparison with similar effects exerted by PGI2. We found that the incubation fluid of deendothelialized aortic samples contained inhibitor activity comparable with that of "intact" samples. The aortic inhibitor had similar properties to PGI2. The aortic inhibitor and PGI2 slightly inhibited light transmission changes of EDTA-PRP following exposure to collagen. However, scanning electron microscopy showed no appreciable difference in platelet adhesion to collagen fibrils. PGI2 and the aortic inhibitor inhibited Kaolin-induced PF-3 availability, but did not prolong the APTT or TT.

The effects of a combined alpha- and beta-blocking drug, labetalol, on some aspects of platelet function.

The effects of the combined alpha- and beta-adrenoreceptor blocking agent labetalol on human blood platelets as estimated by platelet aggregation, platelet count, bleeding time and platelet factor 3 activity were studied in 5 patients. The drug reduced adrenaline-induced platelet aggregation in vitro. However, it did not influence the above platelet function test in therapeutic plasma concentrations in vivo.

The effect of halofenate--free acid on aggregation--the release reaction, coagulant activity, and lipid metabolism of human platelets.

Halofenate--free acid (HFA), the major metabolite of the hypolipidemic drug, halofenate, inhibited platelet aggregation induced by collagen and sodium arachidonate and blocked the second phase of aggregation caused by ADP, thrombin and epinephrine in human platelet-rich plasma. The aggregation of washed platelets by thrombin and collagen was also blocked. HFA also inhibited the release by thrombin and collagen of 5-hydroxytryptamine from dense granules of platelets and the release by thrombin of beta-glucuronidase from platelet alpha-granules. These inhibitory effects were concentration and time-dependent. HFA decreased platelet factor 3 activity by 31% and also inhibited the incorporation of 14C-acetate and U-14C-glucose into platelet lipids by 89% and 56% respectively. Thrombin-induced lipid peroxidation and prostaglandin formation was investigated by measuring the by-product malonyldialdehyde, and this was found to be inhibited by HFA. It is suggested that the effect of HFA on aggregation is attributable to inhibition of the release reaction which may in turn be a consequence of the effects of the drug on platelet lipid synthesis.

The platelet reactivity of vascular graft prostheses: an in vitro model to test the effect of preclotting.

An in vitro perfusion system was used to study the platelet reactivity of the following vascular graft materials when tested with human blood: expanded polytetrafluoroethylene (ePTFE), crimped Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC). These materials were simultaneously compared to silicone rubber (SR) using an identical perfusion circuit with the same donor's blood. All vascular graft materials tested in this in vitro perfusion system caused some degree of platelet activation as shown by a decrease in platelet count, an increase in platelet factor 3 activity, elevation of plasma levels of both platelet factor 4 and beta-thromboglobulin and decreased platelet aggregability. The observed platelet activation was striking for Dacron and especially preclotted Dacron, with ePTFE showing low levels of platelet activation. Platelet activation by Dacron was initially rapid and then levelled off, whereas the platelet activation with preclotted Dacron began more slowly, but reached much greater levels after three hours of in vitro perfusion.

Anti-drug-related antibodies in nonthrombocytopenic cardiac patients.

Drug-related antibodies (quinidine, hydrochlorothiazide, and digoxin) were studied using the 51Cr platelet lysis test and a recently introduced simplified platelet factor 3 assay in a total of 109 patients admitted to a coronary care unit. Quinidine antibodies were found in 16 (14.7%), hydrochlorothiazide antibodies in six (5.5%) and digoxin antibodies in two (1.8%) of 109 patients. All patients had normal hematologic data, including platelet counts. There was a significantly high incidence of quinidine antibodies in male patients (p less than 0.02), despite the previous report that quinidine-induced purpura was seen predominantly in female patients. Ten patients (9.2%) had antiplatelet antibodies. The simplified platelet factor 3 assay can easily be done in most laboratories and appears as sensitive as the 51Cr platelet lysis test. The high incidence of drug-related or antiplatelet antibodies in hematologically asymptomatic patients may indicate the presence of an insidious, compensated thrombolytic state in many patients.

Platelet sodium-proton exchanger and phospholipid-dependent procoagulant activity in patients with type 2 diabetes.

Platelet Na(+)/H(+) exchanger (NHE) activity, phospholipid-dependent thrombin generation, and platelet factor 3 (PF3) availability were measured in 83 type 2 diabetics and in 40 age- and sex-matched healthy subjects. Na(+)/H(+) exchanger activity was significantly increased in diabetic patients in comparison to the controls (kappa = 4.29 +/- 0.71 x 10(-3) x s(-1) v 3.21 +/- 0.64 x 10(-3) x s(-1), P <.00001). However, there was no significant difference between subjects with (kappa = 4.28 +/- 0.75 x 10(-3) x s(-1)) and without (kappa = 4.26 +/- 0.32 x10(-3) x s(-1)) arterial hypertension, as well as between patients with normo- and microalbuminuria or overt proteinuria (kappa = 4.26 +/- 0.58 x 10(-3) x s(-1), kappa = 4.47 +/- 0.93 x 10(-3) x s(-1) and kappa = 4.07 +/- 0.38 x10(-3) x s(-1), respectively). Comparatively high NHE activity was observed in the group of patients with hemoglobin A(1c) (HbA(1c)) less than 7.5%. Multiple regression analysis revealed that the factors independently related to platelet Na(+)/H(+) exchanger activity were: total PF3 activity (beta = 0.77, P =.011) and triglyceride (TG) concentration (beta = 0.44, P =.039). Phospholipid-dependent thrombin generation and PF3 availability were also enhanced in all plasma fractions of diabetic patients, especially in platelet-poor plasma (PPP) and platelet-free plasma (PFP) (P <.0001 and P <.00001, respectively). There was a positive correlation between NHE activity and thrombin generation, as well as with PF3 availability in all plasma fractions. Our results suggest that enhanced platelet Na(+)/H(+) exchanger activity associated with raised phospholipid-dependent procoagulant activity may increase the risk of vascular damage in type 2 diabetic patients.

Clinical pharmacology studies with indobufen (K 3920): inhibitor of platelet aggregation.

When given to 12 subjects at single oral doses of 100 and 300 mg, indobufen caused clear-cut, dose-dependent, reversible inhibition of epinephrine- and collagen-induced platelet aggregation. Platelet factor 3 availability and platelet factor 4 release were not affected by the lower dose but were markedly reduced by the 300-mg dose. Bleeding time was slightly influenced by 100 mg, and 300 mg had a more pronounced effect. Indobufen 300 mg was also intravenously injected to five subjects. When washed platelets obtained before indobufen were resuspended in plasma obtained after indobufen, aggregation was inhibited. This was not the case when washed platelets obtained after indobufen were resuspended in plasma obtained before indobufen. These experiments indicate tha indobufen causes reversible inhibition of platelet functions unlike the effect of acetylsalicylic acid.

Platelet factor 3 in plasma fractions: its relation to microparticle size and thromboses.

Platelet factor 3 (PF3) was assayed by Russell's viper venom (RVV) in three plasma fractions, platelet-rich plasma (PRP), platelet poor plasma (PPP), and 0.1 microns particle-filtered plasma (PFP), in 42 healthy controls, 34 patients with recent cerebrovascular accidents (CVA) and 28 with recent ischemic events from coronary artery disease (CAD). Platelet microparticles (PMP) were assayed in PPP by flow cytometry. Relative to controls, the RVV clotting times were shortened in all three plasma fractions in both patient groups, p < 0.001. PMP were also elevated in both patient groups, p < 0.001. Linear regression analysis showed that the RVV times of PPP are inversely correlated with PMP, p < 0.005, in patient groups but not in controls. There was no correlation of RVV time with PT, APTT or FIB. After converting RVV times to units of PF3 activity, it could be shown that only about 1/4 of the total PF3 activity was contributed by platelets. The major contribution to the PF3 activity in controls was from microparticles < 0.1 microns but in patients was due mainly to microparticles > 0.1 microns. The RVV time was superior to routine coagulation tests in discriminating thrombotic patients from healthy controls.

A study of hemostasis in ischemic cerebrovascular disease. II. Abnormalities in platelet ADP release, platelet cyclooxygenase regeneration time and platelet factor 3 availability.

Most earlier studies of platelet function in stroke patients have been performed in the acute phase and are hampered by diagnostic insecurity. A sample of totally 67 young adults below the age of 55, with ischemic cerebrovascular disease (TIA and minor stroke) were investigated at a late stage after acute disease and compared to 20 healthy controls. Patients with atherosclerotic signs at cerebral angiography had significantly (p less than 0.05) higher platelet factor 3 availability than angionegative patients. Unexpectedly, female patients compared to male patients had significantly (p less than 0.05) larger ADP-release after stimulation with collagen in vitro. Furthermore, when female patients were compared to female controls a significantly (p less than 0.05) increased platelet factor 3 availability was found. The results indicate that platelets in female patients may have an increased tendency to aggregate in vivo. Patients had significantly (p less than 0.01) shortened platelet cyclooxygenase regeneration half times (PRT). This was correlated to high levels of factor VIII related antigen (r=0.59) and high levels of factor VIII biological activity (r=0.67), indicating that platelets may be consumed by platelet adhesion and mural thrombi formation in abnormal vessel walls. PRT appears to be a reliable method to assess platelet function in vivo and to optimize aspirin dose and dose intervals in the individual.

Effect of oral contraceptive use on platelet prothrombin converting (platelet factor 3) activity.

PIP: The attempt was made to determine if platelets obtained from women using oral contraceptives (OCs) have increased activity in support of Factor Xa-catalyzed prothrombin activation. 9 volumes of human blood were collected into 1 volume of 3.2% sodium citrate solution. Fibrinogen was prepared by the method of Straughn and Wagner. Prothrombin was assayed by the taipan sanke venom method, and antithrombin 3 was assayed as heparin cofactor activity by a modification of the method described by Henriksen and Owen. Subjects, including controls, were women between 20-30 years who were in good health and who had not ingested aspirin or other drugs within 10 days of the beginning of the experiment. Platelets from OC users, compared with controls, promote an increase in both rate and extent of prothrombin activation, with control and test groups falling into non-overlapping populations. The increased prothrombin activation correlates with an increase in antithrombin 3 consumption. Enhanced antithrombin 3 consumption explains the decreased serum antithrombin 3 levels among OC users. Considered with increased platelet lability and an increased tendency to aggregate, the increase in total available platelet factor 3 activity points to a specific role of the platelet in the increased risk of thromboembolic disease associated with OC use.^ieng

Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation.