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.

Comparison of blood product use and costs with use of 3-factor versus 4-factor prothrombin complex concentrate for off-label indications.

PURPOSE: Results of a comparison of blood product use and cost outcomes with use of 3-factor versus 4-factor prothrombin complex concentrate (PCC) for indications other than warfarin reversal are presented. METHODS: Consecutive patients who received 3-factor PPC (PCC3) or 4-factor PCC (PCC4) for non-warfarin-related indications at 2 U.S. hospitals during a 19-month period were identified. The primary outcome was in-hospital blood product use, with a focus on plasma use. Total hemostasis costs, intensive care unit (ICU) and hospital lengths of stay, and other outcomes were evaluated. RESULTS: Indications for PCC3 use (n = 118) or PCC4 use (n = 64) included intraoperative bleeding, nonintraoperative bleeding, coagulopathy of liver disease, and reversal of direct-acting oral anticoagulant effects. The proportion of patients who received plasma was 56.8% with PCC3 use versus 53.1% with PCC4 use (p = 0.643); the corresponding median volumes of plasma received were 638 mL (interquartile range [IQR], 550-1,355 mL) and 656 mL (IQR, 532-1,136 mL), respectively. The median total hemostasis costs were $5,559 (IQR, $3,922-$8,159) with PCC3 use and $7,771 (IQR, $6,366-$9,205) with PCC4 use (p < 0.001). CONCLUSION: PCC3 use and PCC4 use were associated with similar blood product use, ICU length of stay, hospital length of stay, and in-hospital mortality when given for non-warfarin-related indications. However, relative to PCC3 use, PCC4 use was associated with an increase in costs that was primarily due to drug costs.

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.

Operator dependent choice of prostate cancer biopsy has limited impact on a gene signature analysis for the highly expressed genes IGFBP3 and F3 in prostate cancer epithelial cells.

BACKGROUND: Predicting the prognosis of prostate cancer disease through gene expression analysis is receiving increasing interest. In many cases, such analyses are based on formalin-fixed, paraffin embedded (FFPE) core needle biopsy material on which Gleason grading for diagnosis has been conducted. Since each patient typically has multiple biopsy samples, and since Gleason grading is an operator dependent procedure known to be difficult, the impact of the operator's choice of biopsy was evaluated. METHODS: Multiple biopsy samples from 43 patients were evaluated using a previously reported gene signature of IGFBP3, F3 and VGLL3 with potential prognostic value in estimating overall survival at diagnosis of prostate cancer. A four multiplex one-step qRT-PCR test kit, designed and optimized for measuring the signature in FFPE core needle biopsy samples was used. Concordance of gene expression levels between primary and secondary Gleason tumor patterns, as well as benign tissue specimens, was analyzed. RESULTS: The gene expression levels of IGFBP3 and F3 in prostate cancer epithelial cell-containing tissue representing the primary and secondary Gleason patterns were high and consistent, while the low expressed VGLL3 showed more variation in its expression levels. CONCLUSION: The assessment of IGFBP3 and F3 gene expression levels in prostate cancer tissue is independent of Gleason patterns, meaning that the impact of operator's choice of biopsy is low.

Bilateral vitreous hemorrhage in a child due to isolated platelet factor 3 availability defect.

Vitreous hemorrhage is an uncommon cause of decreased vision in children. We report a 3-year-old child with bilateral vitreous hemorrhage secondary to an isolated platelet factor 3 (PF-3) availability defect. Prophylactic infusion of platelet-rich plasma followed by pars plana vitrectomy in both eyes resulted in an excellent surgical and visual outcome. This report emphasizes the importance of exhaustive hematological and platelet function studies in a child presenting with bilateral vitreous hemorrhage and its implications on the management of present and future bleeding episodes.

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.

Significance of platelet activation in sickle cell anaemia.

BACKGROUND: There is increasing evidence suggesting the contribution of platelets in the vaso-occlusive phenomena found in sickle cell anaemia. This study is aimed at using simple, inexpensive parameters to determine the role of platelets in the steady state and vaso-occlusive crisis state of Nigerian sickle cell anaemia patients. METHODS: The circulating platelet aggregate (CPA) ratio, platelet factor-3 availability (PF-3) and platelet counts of 60 adult Nigerian sickle cell anaemia patients were studied. RESULTS: The CPA ratio in the sickle cell anaemia (SCA) patients in steady state (SS) was 0.93 +/- 0.05, 0.89 +/- 0.04 during vaso-occlusive crisis (VOC) and 0.98 +/- 0.02 in the control group (C). The values in the vaso-occlusive crisis and in steady state were significantly lower than in the control group (P < 0.05). PF3 availability in steady state and vaso-occlusive crisis were 29.7 +/- 4.0 secs and 28.4 +/- secs respectively. The times are significantly shorter when compared with the control group with a time of 36.2 +/- 4.3 secs (P < 0.05). There was however no significant difference between the two sickle cell groups. Platelet count was significantly raised in the steady state patients 224.3 +/- 46.3 x 10(9)/L when compared with controls of 196.6 +/- 39.3 x 10(9)/L (P < 0.05). There was a significant fall during VOC to 140.6 +/- 36.3 x 10(9)/L (P < 0.05). The difference between the two sickle cell groups is significant (P < 0.05). CONCLUSION: This study indicates varying degrees of partial activation of platelets in vivo in the steady state and vaso-occlusive crisis state of sickle cell anaemia. It support's a contribution of platelet to the vascular occlusion that underlies much of the morbidity in the disease.

[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.

Platelet-derived microparticles stimulate proliferation of granulocyte-macrophage progenitor cells from umbilical cord blood / 中国实验血液学杂志

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.

Haemarthroses after total knee arthroplasty caused by an isolated platelet factor 3 availability defect.

We present seven patients with recurrent haemarthroses after total knee arthroplasty, caused by an inherent platelet function defect. These patients developed painful knee swelling, persistent bleeding and/or wound breakdown, a platelet factor 3 availability defect being identified in all cases. Surgical exploration, with joint debridement, lavage and synovectomy, was performed in four patients who did not improve with conservative therapy. Histopathological examination of synovium revealed a focal synovial reaction with histiocytic infiltration, and occasional foreign-body giant cells. One patient required an early revision because of aseptic loosening of their tibial component. The condition was treated by single-donor platelet transfusions with good results. The diagnosis, management, and relevance of this disorder are discussed.

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.

[Study on effects of Erigeron breviscapus extract on anticoagulation].

OBJECTIVE: To study the effect of Erigeron breviscapus extract on anticoagulation. METHOD: Coagulation time (CT), prothrombin time (PT), platelet factor III (PF3) and euglobulin lysis time (ELT) have been used to evaluate the anticoagulation activities of Erigeron breviscapus extract. RESULT: Erigeron breviscapus extract significantly delayed CT and PT, inhibited the activity of PF3, and decreased ELT. CONCLUSION: Erigeron breviscapus extract plays an role in anticoagulation by inhibiting PF3 and prothrombin V. It can also enhance the activity of fibrimolysis.

Different effects of various anti-GPIIb-IIIa agents on shear-induced platelet activation and expression of procoagulant activity.

Inhibitors of the platelet glycoprotein (GP)IIb-IIIa receptor (integrin alphaIIbbeta3) reduce acute thrombotic events in patients with coronary artery disease. To characterize the mechanism of action of these drugs, we evaluated the effects of different GPIIb-IIIa antagonists on shear-induced platelet aggregation, activation, and the expression of procoagulant activity. Samples of platelet-rich plasma from 16 volunteers were exposed to the shear rate of 10 800 s-1 for 6 min in an optically modified cone-plate viscometer. Abciximab, tirofiban and eptifibatide inhibited aggregation to a similar extent (mean +/- SD: 74.1 +/- 8.5%, 69.5 +/- 13.6%, 65.6 +/- 17.0%, respectively), but only abciximab inhibited significantly microparticle release associated with shear-induced platelet activation (64.4 +/- 13.6%, P = 2.2 x 10-7; tirofiban = 20.0 +/- 23.4%; eptifibatide = 23.9 +/- 17.4%). P-selectin platelet surface translocation was also strongly inhibited by abciximab, weakly by eptifibatide, but not by tirofiban. The addition of anti-alphavbeta3 to tirofiban enhanced the inhibiting effects on shear-induced P-selectin translocation and microparticle release. Shearing of platelet-rich plasma shortened the re-calcification clotting time after addition of kaolin from 106.9 +/- 14.3 to 94.2 +/- 10.7 s (mean +/- SD; P = 0.0013). This effect, which is mediated by the appearance of procoagulant phospholipids on the surface of sheared platelets and microparticles, was prevented by abciximab and by the combination of tirofiban and anti-alphavbeta3, but not by tirofiban alone or eptifibatide. The ability to inhibit shear-induced platelet activation, as evidenced by microparticle release and P-selectin surface translocation as well as the expression of procoagulant activity, differentiates the effects of anti-GPIIb-IIIa agents, which may explain the distinct antithrombotic efficacy of the agents.

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.

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.