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.

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.

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.

[Factors influencing platelet procoagulant activity in type II diabetic patients. The role of sodium-proton exchange]. / Czynniki regulujace aktywnosc prokoagulacyjna plytek krwi u pacjentów z cukrzyca typu 2. Rola antyporterów sodowo-protonowych.

The aim of our study was the estimation of platelet sodium-proton exchanger activity and platelet pro-coagulant activity, expressed as the availability of platelet factor 3 (PF3) and thrombin generation, in 83 type 2 diabetic patients (mean age 56.7 +/- 7.8 years) and 40 healthy subjects (mean age 54.4 +/- 6.2 years). Thrombin generation was measured in platelet rich plasma, using a chromogenic substrate S-2238. The availability of PF3 was estimated in platelet rich plasma, platelet poor plasma and platelet filtrated plasma, to assess procoagulant activity connected with platelets and cell derived microparticles, shedding upon activation (according to Jy and Horstman). The activity of platelet Na+/H+ exchanger was measured using an optical swelling assay. We found that the activity of PF3 and phospholipid dependent thrombin generation were significantly higher in diabetic patients, irrespective of their vascular complications and metabolic control. The highest increase of PF3 activity was observed in platelet poor (p < 0.0001) and platelet filtrated plasma (p < 0.000001). Na+/H+ exchange rate was significantly higher in diabetic patients in comparison to the controls (4.29 +/- 0.71 x 10(-3)/s vs 3.21 +/- 0.64 x 10(-3)/s, p < 0.00001). There was also a positive correlation between Na+/H+ exchanger activity and PF3 activity in all plasma fractions. Our results suggest that increased thrombin generation, enhanced platelet Na+/H+ exchanger activity and raised PF3 availability, connected mainly with cell derived microparticles, may enhance the risk of vascular damage in type 2 diabetic patients.

Generation of annexin V-positive platelets and shedding of microparticles with stimulus-dependent procoagulant activity during storage of platelets at 4 degrees C.

BACKGROUND: The ability of propyl gallate to activate platelet factor 3 has been determined through the activated partial thromboplastin time, but its effect on phosphatidylserine has not been established. STUDY DESIGN AND METHODS: A novel platelet activator, propyl gallate, was introduced to a study of platelets stored at 4 degrees C. The effects of storage on platelet coagulation activity, on phosphatidylserine, and on the shedding of activated and activable membrane particles (microparticles) were examined by activated plasma clotting time, and the effect on annexin V binding was examined by gated flow cytometry. The ratios of annexin V binding and microparticle shedding in stored platelet samples were compared with those in fresh platelets stimulated with propyl gallate. RESULTS: Microparticle shedding by stored platelets compensated for the diminished procoagulant potential of intact platelets (shown as the total propyl gallate-dependent platelet factor 3 activity), which did not change during prolonged (20-day) storage, but levels of phosphatidylserine confined to microparticles increased dramatically as platelet counts fell. Both annexin V binding and microparticle shedding increased spontaneously with storage and artificially with propyl gallate stimulation. However, at the same level of annexin V binding, stored platelets shed more microparticles than did fresh platelets stimulated with propyl gallate. CONCLUSION: Propyl gallate induces platelet procoagulant activity and annexin V binding. Stored platelets differ from fresh platelets in a lower reactivity to propyl gallate activation and a higher rate of microparticle shedding.

Clinico-haematological profile of isolated PF3 availability defect: therapeutic potential of soya bean--a pilot study.

Isolated platelet factor 3 (PF3) availability defect has been observed to be a common platelet functional disorder (PFD) in the Department of Haematology, All India Institute of Medical Sciences, New Delhi, India. One hundred and thirty-two patients were diagnosed to have this defect based on the presence of reduced PF3 availability, normal platelet aggregation with ADP, collagen, adrenaline, ristocetin, and arachidonic acid and normal PF3 content. PF3 availability was evaluated by measurement of Russel viper venom time (RVVT) on the patient's platelet-rich plasma (PRP) after incubation with ADP for 20 min. An RVVT value >19.0 s was considered diagnostic of reduced PF3 availability in patients with normal prothrombin and activated partial thromboplastin times. Isolated PF3 availability defect occurred in patients with ages between 2 and 65 yr and had a female preponderance (M:F=1:2). One fifth of the patients had a positive family history of similar mild bleeding diathesis, indicating an autosomal dominant pattern of inheritance. All patients presented with mild bleeding manifestations, the commonest symptom being appearance of recurrent ecchymotic spots. In females, menorrhagia was the commonest symptom. A pilot study was conducted on 45 patients to evaluate the therapeutic role of oral soya bean (50 g/d). The clinical response was evaluated after 3 months. Soya therapy resulted in disappearance of bleeding problems in 5 patients and reduction in frequency and severity of bleeding in 26 patients. A repeat PF3 availability test after 3 months of therapy showed complete correction in 4 and partial correction in 12 patients. It is evident from McNemer's test that both the clinical and the laboratory parameters (PF3 availability) showed a similar response to soya therapy (p>0.05). Pre-soya therapy mean PF3 availability values differ significantly from those after soya therapy (p<0.01). Thus, soya bean appears to have a therapeutic potential in isolated PF3 availability defect.

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.

Influence of indobufen on occlusion of arteriovenous fistulas and some platelet activity in intermittent peritoneal dialysis patients.

Thrombotic complications constitute a significant problem connected with maintaining arteriovenous fistulas (A-V) for a long time. It has been established that platelets play an important role in the development of thrombosis in high flow systems. Aspirin and dipyridamole do not decrease the frequency of shunt thrombosis. Some of the more recently synthetised antiplatelet drugs (i.e. indobufen, 2-p-oxo-isoindolinyl-phenyl-butyric acid) could be promising in the prevention of such complications. The study group consisted of 40 patients in the terminal stage of renal failure treated with intermittent peritoneal dialysis (IPD). The A-V fistulas were formed by the same surgeon anesthetist team and this allowed for the elimination of technical errors. All patients were divided into two groups. Group I received indobufen at the dose of 2 x 100 mg/24 h orally. Group II received no antiplatelet treatment. The therapy started 24 h before A-V formation. The treatment was continued for 3 weeks. The following tests of platelet function were performed before indobufen therapy, after 9 h and 3 weeks of treatment: ADP and adrenaline induced platelet aggregation, platelet circulating aggregates, MDA level, platelet factor 3 and 4 and bleeding time. During indobufen treatment only a significant decrease in ADP induced aggregation was observed. No prolongation of the bleeding time was noted. No case of fistula thrombosis in indobufen group was observed. This complication, however, appeared in 3 patients (15%) of the control group (without antiplatelet therapy).

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.

Platelet function studies in Indian kala-azar.

Platelet function studies were conducted on 25 parasitologically positive cases of Indian kala-azar and 25 age and sex matched healthy controls. Ninety-two per cent of patients had thrombocytopenia of variable degree; in 44% of patients, platelets were less than 60,000 mm-3. The platelet adhesive index was less than 30% in 70% of patients with kala-azar (normal 31-60%). Platelet aggregation time with ADP and adrenaline was abnormally prolonged compared to the controls. Platelet factor III availability was poor in 40% of cases. There was a fair degree of correlation between platelet adhesiveness and platelet factor III availability in these patients: 50% of patients with poor platelet adhesiveness showed reduced platelet factor III availability.

[Distribution of phospholipids in platelet membranes and platelet factor 3 availability in patients with chronic kidney failure]. / Rozmieszczenie fosfolipidów blonowych krwinek plytkowych a dostepnosc 3 czynnika plytkowego u chorych z przewlekla niewydolnoscia nerek.

Patients with end-stage renal failure (ESRF) have a defect of haemostasis mainly caused by disturbances of platelet vessel wall interactions. An essential part in platelet function is played by the membrane phospholipids. The aim of this study was to estimate phospholipid distribution in platelet membrane and to evaluate the correlation between this distribution and PF3 availability. 18 non-dialysed (group I) and 21 chronically haemodialysed patients (group II) were studied. Phospholipid distribution was determined using TNBS tracer by the Vale method, and by phospholipase hydrolysis according to the modified Chap method. PF3 availability was estimated by the Saleem method. In group I membrane phospholipid distribution was unchanged as compared with control group, in this group decreased PF3 availability was also shown. In group II higher content of phosphatidylserine (PS) and phosphatidylethanolamine (PE) at the outer surface of platelet membrane was observed as compared with the healthy persons. In this group increased PF3 availability correlated with PS and PE exposition was also noted. It may reflects platelet activation in these patients. The different PF3 availability in non-dialysed and chronically haemodialysed patients suggests various pathogenesis of haemostasis disturbances in their patients.

Effect of red blood cells from patients with sickle cell disease on platelet factor 3 release.

Red cells from two SS genotype and two SA genotype patients were sampled. When the samples were deoxygenated and mixed with platelet-rich plasma, they caused the release of platelet factor 3 as recorded in a coagulometer. This phenomenon was not present in control blood samples from normal individuals. Membrane changes in abnormal red cells during hypoxia may be responsible in part for platelet activation and its role in vasoocclusive crisis. Vasoocclusive crisis could be prevented by increasing the red cell membrane fluidity and inhibiting the platelet aggregation with pentoxifylline.

Vesiculation of platelets during in vitro aging.

Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

Complement components in neonatal sepsis.

Complement components C3, C1q, factor B and breakdown products of C3, i.e. C3c and C3d, were evaluated in the diagnosis and prognosis of sepsis in 24 neonates with proven sepsis. The complement components were measured by electroimmunodiffusion and breakdown products by counterimmunoelectrophoresis (CIEP). The babies with sepsis were found to have decreased levels of C1q and factor B as compared with suitably matched healthy controls. No statistically significant depression was observed in C3 levels of infected babies. However, breakdown products of C3, i.e. C3c and C3d, were detected in 58.3% of these babies. The breakdown products of C3 were not present in any of the healthy controls. The degree of depression of complement components was of no prognostic significance in neonatal sepsis.

The effect of carbamoylpiperidine and nipecotoylpiperazine analogs on ADP-stimulated human thrombocyte aggregation and platelet factor 3 availability.

There is a striking congruence between the inhibitory effects of three synthetic entities on ADP-induced (i) human blood platelet aggregation and (ii) platelet factor 3 availability as evidenced by prolonged 'Stypven time'. The pronounced parallel between each compound's potency in inhibiting aggregation (e.g. IA 48.9 +/- 1.3, S.E., %; n = 16) and in impeding platelet factor 3 availability (e.g. IPF-3av 42.3 +/- 2.5, S.E., %; n = 12), determined concurrently in platelet-rich plasma of four different donors, further substantiates that the antiplatelet activity of our carbamoylpiperidine and nipecotoylpiperazine congeners is exerted through their interaction with anionic phospholipids.

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.