Multimeric analysis in diagnosis of vWD variants in Indians.

von Willebrand disease is a common inherited bleeding disorder and the problem is undefined in developing countries due to limitation of its diagnostic facilities. The aim of the study was to diagnose vWD in patients with history of muco - cutaneous bleeding and characterization into its variants by multimeric analysis. 224 patients presenting with history of muco - cutaneous bleeding were selected. In all patients, platelet count, BT, PT, APTT, PF3 availability, clot solubility and factor VIII assay were done. Diagnosis of vWD was confirmed by RIPA, vWF: Ag, and vWF: RCo and its sub-characterization was done by multimeric analysis. 64 patients were diagnosed to have vWD. Of these, 21.9% were of type 1 vWD, 43.7% type 2 vWD, 1.6% acquired vWD and 32.8% type 3 vWD. By multimeric analysis, 2 patients had supranormal HMW multimers and two patients had normal distribution of vWF multimers were diagnosed as type 2M 'Vicenza'; and type 2M vWD respectively. It is concluded, that vWD is not an uncommon condition amongst Indian population.

Activated platelet-derived microparticles in thalassaemia.

Thromboembolic complications have been documented in thalassaemia patients. The aggregability of abnormal red blood cells and the high level of membrane-derived microparticles (MPs) stemming from blood cells are thought to be responsible for the associated thrombotic risk. We investigated the number of MPs, their cellular origin and their procoagulant properties in beta-thalassaemia. Fresh whole blood was simultaneously stained for annexin V, cellular antigens and the known density beads. The procoagulant properties of these phosphatidylserine (PS)-bearing MPs were also measured by assessing the platelet factor-3-like activity in the blood. Flow cytometric results showed that splenectomised beta-thalassaemia/HbE patients had significantly higher levels of PS-bearing MPs than non-splenectomised beta-thalassaemia/HbE patients and normal individuals (P < 0.0001). There was a good correlation between PS-bearing MPs and PS-bearing platelets, reflecting the existence of chronic platelet activation in beta-thalassaemia/HbE patients (r(s) = 0.511, P < 0.001). The cellular origin of PS-bearing MPs showed mostly activated-platelet origin with adhesion (CD41a/CD62P/CD36). Moreover, the platelet procoagulant activity was higher in splenectomised beta-thalassaemia/HbE patients when compared with non-splenectomised (P < 0.05) and normal individuals (P < 0.01), and the amount correlated with PS-bearing MPs (rs = 0.560, P < 0.001). These findings suggest that MPs originate from activated platelets with a potential to aggravate thrombotic events when the numbers are excessive, as is commonly seen in splenectomised beta-thalassaemia/HbE patients.

[Platelet-derived microparticles stimulate proliferation of granulocyte-macrophage progenitor cells from umbilical cord blood].

This study was aimed to investigate the effect of platelet-derived microparticles (PMP) on stimulating the proliferation of granulocyte-macrophage progenitors (CFU-GM) from umbilical cord blood. Different concentrations of thrombin were adopted to activate the platelets for releasing PMP. Flow cytometry was adopted to evaluate the efficiencies of different concentrations of thrombin to produce PMP. Umbilical cord blood mononuclear cells (MNC) were obtained from healthy donors and the MNC were isolated by Ficoll density gradient centrifugation. MNC were cultured in 2.7% methylcellulose containing different concentration of PMP and colonies were counted under an inverted microscope after 7 days. The result showed that the release rate of PMP activated by 2.0, 1.5, 1.0 and 0.5 U/ml thrombin were 28.7%, 47.7%, 50.1% and 43.9% respectively. The PMP enhanced colony formation in dose-dependent manner. The number of colonies per 2 x 10(5) MNCs in groups of PMP at different concentrations (10, 50 and 100 microg/ml) were 119.8 +/- 32.2,142.8 +/- 45.2 and 180.8 +/- 85.1 respectively. The number of colonies in the groups of PMP at 100 microg/ml and 50 microg/ml were statistically significant when compared with control group (103.0 +/- 24.8) (P < 0.05). The number of colonies per 2 x 10(5) MNC in the group of PMP (10 microg/ml) was 119.8 +/- 32.2 which was higher than that in control group, but there was no statistical significance between two groups. It is concluded that platelet activated with 1.0 U/ml thrombin can get the best release efficiency of PMP and PMP can enhance the proliferation of granulocyte-macrophage progenitor cells of umbilical cord blood.

A hereditary bleeding disorder of dogs caused by a lack of platelet procoagulant activity.

We have discovered a novel canine hereditary bleeding disorder with the characteristic features of Scott syndrome, a rare defect of platelet procoagulant activity. Affected dogs were from a single, inbred colony and experienced clinical signs of epistaxis, hyphema, intramuscular hematoma, and prolonged bleeding with cutaneous bruising after surgery. The hemostatic abnormalities identified were restricted to tests of platelet procoagulant activity, whereas platelet count, platelet morphology under light microscopy, bleeding time, clot retraction, and platelet aggregation and secretion in response to thrombin, collagen, and adenosine diphosphate stimulation were all within normal limits. Washed platelets from the affected dogs demonstrated approximately twice normal clotting times in a platelet factor 3 availability assay and, in a prothrombinase assay, generated only background levels of thrombin in response to calcium ionophore, thrombin, or combined thrombin plus collagen stimulation. While platelet phospholipid content was normal, flow cytometric analyses revealed diminished phosphatidylserine exposure and a failure of microvesiculation in response to calcium ionophore, thrombin, and collagen stimulation. Pedigree studies indicate a likely homozygous recessive inheritance pattern of the defect. These findings confirm the importance of platelet procoagulant activity for in vivo hemostasis and provide a large animal model for studying agonist-induced signal transduction, calcium mobilization, and effector pathways involved in the late platelet response of transmembrane phospholipid movement and membrane vesiculation.

Increased platelet factor 3 activity in Plasmodium falciparum malaria.

Platelet count and platelet factor 3 (pf 3) availability were determined in twenty five febrile subjects with no parasitologic Plasmodium falciparum malaria and in thirty eight febrile subjects with parasitologic malaria. Eighteen age and sex matched afebrile subjects without parasitologic malaria served as controls. Platelet counts were significantly lower in the malaria patients (p < 0.01) and in the non-parasitologic but febrile subjects (p < 0.01) than the control group. Platelet factor 3 activity was significantly higher in the febrile group (p < 0.01) than in the control subjects. Parasite density did not correlate either with platelet count or with platelet factor 3 clotting time.

Platelet count and platelet factor 3 (PF-3) availability in sickle cell disease.

In view of the effect of red blood cell membrane on platelet factor 3 release in sickle cell disease, platelet count and platelet factor 3 (PF-3) availability were measured in 30 sickle cell patients in a stable state and 15 with thrombocytosis, and these were compared with 60 non-sickle cell individuals as controls. There were no significant differences in platelet counts, but there was a slightly higher PF-3 availability in sickle cell patients not in crisis than in the controls (P < 0.025). Sickle cell patients with thrombocytosis made PF-3 available more readily than did the control individuals and sickle cell patients not in crisis (P < 0.001). We conclude that sickle cell patients are capable of making platelet factor 3 readily available for coagulation activation, and this may play a decisive role in vaso-occlusive crisis.

Influence of teniposide on platelet functions in vitro.

Teniposide added to citrated platelet-rich plasma reduced platelet aggregation induced by collagen, but did not interfere with ADP-induced aggregation. The availability of platelet factor 3 was decreased irrespective of inducer. Reptilase clot retraction induced by ADP or collagen was reduced. Teniposide did not interfere with platelet adhesion to glass. It did not release lactic dehydrogenase from the cytoplasm of platelets. Coagulation factors were not affected.

Action of 2-acetoxy-trifluoromethylbenzoic acid (Triflusal) on platelet function after repeated oral administration in man: a pharmacological clinical study.

The efficacy and safety of Triflusal capsules given to patients at thrombogenetic risk because of platelet hyperaggregation were investigated in a controlled study involving 15 patients (9 males and 6 females, mean age 65.7 years) who were given 300 mg/day of Triflusal during the first 5 days and 600 mg/day during the following 5 days. Subsequently, after 7 days of wash-out all these patients received placebo for 10 days, one capsule for the first 5 days and 2 capsules for the following 5 days. The platelet antiaggregant activity of the drug was evaluated by means of Born's platelet activation test. Specific tests were also made to assess the effect of this substance on platelet release and the coagulation system. The safety was evaluated by measuring the most important clinical chemistry and clinical haematology indexes of haematopoietic, hepatic, renal and metabolic functions. Arterial blood pressure and heart rate values were also recorded. All the 15 patients completed the study. It was found that the Triflusal treatment led to a significant mean reduction of the indexes chosen as markers of thrombophilia or platelet hyperaggregation in vivo. It did not affect the normal haemostatic-coagulation process and was well tolerated by the patients. The subsequent placebo treatment did not induce any platelet antiaggregant effects.

Hemostatic profile in neonatal septicemia.

Hemostatic profile was studied in 25 full term non-asphyxiated neonates with blood culture-proven septicemia. Observations were compared with that of 25 healthy, non-asphyxiated, full term, birth weight and age-matched controls. Detailed coagulation tests & platelet studies were done in each of the 50 neonates by standard techniques. Hemostatic defects occurred in 96% of the septicemic neonates and none in the control group irrespective of the occurrence of clinical bleeding. The coagulation tests were deranged in 805 and platelet function tests in 92% of patients. These tests were significantly deranged in septicemic neonates as compared to control group.

[The estimation of platelet factor 3 activity in horses, cattle, sheep and pigs by the use of synthetic chromogenic tripeptide substrates]. / Oznaczanie aktywnosci czynnika plytkowego 3 u koni, bydla, owiec i swin przy uzyciu chromogennego substratu trójpeptydowego.

The aim of this study was to investigate the platelet factor 3 activity in platelet-rich plasma of horse, cattle, sheep and pig, by the use of chromogenic tripeptide substrate H-D-Phenylalanyl-Pipecolyl-Arginyl-p-nitroanilide. Among species examined the highest activity of this factor was stated in pig, the lowest one in sheep. Chromogenic substrate test was 10 times more sensitive that Stypven clotting time test. Thus, the use of chromogenic tripeptide substrate is fully valuable in platelet factor 3 activity estimation in domestic animals.

Hemorheologic effects of Meclomen including effect in malignant melanoma.

Sodium Meclofenamate, (Meclomen) (M) is a non-steroidal anti-inflammatory drug, which inhibits both cyclooxygenases and lipooxygenases of the arachidonic acid cascade (AA). (M) inhibited adenosine diphosphosphate (ADP) induced platelet aggregation, arachidonic acid (AA) induced platelet aggregation and collagen induced platelet aggregation. (M) inhibited platelet factor 3 availability when measured by Stypven clotting time. Red cells from patients with advanced malignancies and normal red cells stored in blood banks exhibited loss of deformability. (M) enhanced red cell deformability in these blood samples.

Lack of significant effect of therapeutic propranolol on measurable platelet function in healthy subjects.

Propranolol, a non-selective beta blocker, was administered orally in therapeutic doses. The effects of a single dose (160 mg) and one week's treatment (80 mg twice a day) on platelet function were compared in healthy young subjects. There were no significant changes in circulating platelet aggregates, template bleeding time, platelet factor 3 availability and thromboxane beta 2 (TX beta 2) generation. Platelet aggregation responses as assessed by angle of slope and maximal percentage aggregation (all agonists) and lag phase (collagen) showed no changes of biological importance, although minor changes reaching significance were observed with some agonists. These findings suggest that propranolol does not significantly affect platelet function when taken at doses commonly encountered in clinical practice.

Coagulation activities of plasma microparticles.

An evaluation of the effect of plasma microparticles (MP) on in vitro coagulation has been undertaken using platelet rich (PRP), platelet poor (PPP) and platelet free (PFP) plasmas prepared by differential centrifugation. MP provide coagulant material which shortens the activated partial thromboplastin time (APTT) and dilute simplastin time (DSTT) which is different from that contributed by commercial phospholipid preparations. The amount of platelet factor three (PF3) available in plasma is directly correlated with the centrifugal force used in its preparation and is present in large amounts in the MP pellet remaining after preparation of PFP. Factor VIII (F.VIII:C) and von Willebrand factor (vWf) were associated with the MP fraction but could be separated from MP on sucrose gradients. The effect of MP on the APTT was independent of the F.VIII:C/vWf and was not solely due to their PF3 content. Plasma prepared for routine coagulation assays contains MP which contribute to the APTT and DSTT and should be considered in their assessment. High speed centrifugation of plasma reduces the F.VIII:C/vWf:Ag/RCoF levels and this may contribute to losses of these proteins during preparative procedures utilising high speed centrifugation.

Effects of nicotine on platelet function.

In non-smokers, only high concentrations of nicotine (10 mM) caused platelet aggregation in platelet-rich plasma and release of 5-hydroxytryptamine (5-HT). Both responses to ADP and 5-HT were enhanced at 1 and 10 mM nicotine while they were inhibited to collagen, ristocetin, adrenaline and arachidonic acid. Lower concentrations of nicotine had no effect with any agent other than 5-HT, with which a variable enhancement of 5-HT-induced aggregation was observed. Uptake of 14C-5-HT was inhibited by nicotine at 100 microM or higher while platelet factor 3 availability was unaffected. Thus it is unlikely that direct effects of nicotine on platelets are responsible for smoking-related changes in platelet reactivity.