A novel variant on chromosome 7q22.3 associated with mean platelet volume, counts, and function

Mean platelet volume (MPV) and platelet count (PLT) are highly heritable and tightly regulated traits. We performed a genome-wide association study for MPV and identified one SNP, rs342293, as having highly significant and reproducible association with MPV (per-G allele effect 0.016 +/- 0.001 log fL; P < 1.08 x 10(-24)) and PLT (per-G effect -4.55 +/- 0.80 10(9)/L; P < 7.19 x 10(-8)) in 8586 healthy subjects. Whole-genome expression analysis in the 1-MB region showed a significant association with platelet transcript levels for PIK3CG (n = 35; P = .047). The G allele at rs342293 was also associated with decreased binding of annexin V to platelets activated with collagen-related peptide (n = 84; P = .003). The region 7q22.3 identifies the first QTL influencing platelet volume, counts, and function in healthy subjects. Notably, the association signal maps to a chromosome region implicated in myeloid malignancies, indicating this site as an important regulatory site for hematopoiesis. The identification of loci regulating MPV by this and other studies will increase our insight in the processes of megakaryopoiesis and proplatelet formation, and it may aid the identification of genes that are somatically mutated in essential thrombocytosis. (Blood. 2009; 113: 3831-3837)

The effect of exercise and training status on platelet activation: do cocoa polyphenols play a role?

Sedentary and trained men respond differently to the same intensity of exercise, this is probably related to their platelet reactivity and antioxidant capacity. There is growing interest in the utilization of antioxidant-rich plant extracts as dietary food supplements. The aim of this study was to investigate the effect of an acute bout of sub maximal exercise on platelet count and differential response of platelet activation in trained and sedentary subjects and to observe if cocoa polyphenols reverse the effect of exercise on platelet function. The practical significance of this study was that many sedentary people engage in occasional strenuous exercise that may predispose them to risk of heart disease. Fasting blood samples were collected from 16 male subjects, pre and post 1-h cycling exercise at 70% of maximal aerobic power (VO2max) before and after consumption of cocoa or placebo. Agonist stimulated citrated whole blood was utilized for measuring platelet aggregation, adenosine triphosphate (ATP) release and platelet activation. Baseline platelet count (221 ± 33 times 109/L) and ATP release (1.4 ± 0.6 nmol) increased significantly (P < 0.05) after exercise in all subjects. Baseline platelet numbers in the trained were higher (P < 0.05) than in the sedentary (235 ± 37 vs. 208 ± 34 times 109/L), where as platelet activation in trained was lower (P < 0.05) than sedentary (51 ± 6 vs. 59 ± 5%). Seven days of cocoa polyphenol supplementation had little effect on any of the parameters measured. We conclude that trained subjects show decreased activation of stimulated platelets when compared to the sedentary subjects and short-term cocoa polyphenol supplementation did not decrease platelet activity in response to exercise independent of prior training status.

Immune thrombocytopenic purpura (ITP) and breast cancer. Does adjuvant therapy for breast cancer improve platelet counts in ITP?

Although both breast cancer and immune thrombocytopenic purpura (ITP) are common conditions, the simultaneous coexistence of these two diseases is rare. ITP is an autoimmune disease in which the presence of autoantibodies against platelets results in splenic sequestration and thrombocytopenia that may be associated with lymphoid neoplasms [1]. Except for an observational case series of 10 patients [2], only a few individual case reports of ITP coinciding with breast cancer have been reported [3–8]. We are reporting two cases with simultaneous confirmed ITP and breast cancer. The platelet counts in both women have improved during adjuvant breast cancer chemotherapy.

Platelet aggregation and TGF-beta(1) plasma levels in pregnant women with preeclampsia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of platelet number and function and fibrinogen level in patients bitten by snakes of the Bothrops genus.

Platelet function and plasma fibrinogen levels were evaluated in 14 patients, 10 males and 4 females, aged 13-59 years bitten by Bothrops genus snakes. There was a statistical difference (p < 0.05) among plasma fibrinogen levels evaluated 24 and 48 hours after envenomation. There was a tendency towards normalization after 48 hours of treatment. The low platelet number was clear in 24-48 hour evaluations with a tendency towards normalization after 48 hours of treatment (p < 0.05). When platelet function was stimulated by collagen and epinephrine, it appeared to be within normal values. On the other hand, when it was stimulated by adenosine diphosphate (ADP), platelet function was hypoaggregated by a single micromol concentration until 48 hours after treatment. At a 3 micromol concentration, there were alterations only before specific treatment (p < 0.05). Fibrinogen levels and fibrin degradation product (FDP) levels appeared to be altered in 83.33% of patients evaluated. The authors suggest that platelet hypoaggregation is related to decreased fibrinogen and increased FDP levels.

Standardization of platelet parameters determination in blood samples collected with EDTA

The Abbott Cell-Dyn 3000 automated hematological analyzer prepares a histogram of platelet volume, which is measured by a technique using electrical impedance, inferring from its platelet count (PLT), mean platelet volume (MPV) and platelet distribution width (PDW). This equipment also calculates the plateletcrit (PCT). These platelet parameters may be important to evaluate platelet function, but they require standardization, because platelets swell when in contact with ethylenediaminetetra-acetic acid salts, hence an increase in blood sample storage time produces artificially increased results. To assess the effect of storage time on MPV, PLT, PDW and PCT, blood samples from 23 sickle cell anemia patients during steady state (Group I) and 50 from healthy controls (Group II) were placed in Vacutainer® tubes with dipotassium ethylenediaminetetra-acetic acid and measured over a period of 1440 minutes (24 h) at the following times: immediately after the venipuncture (time 0), 15, 30, 60, 120, 240, 360, 480 and 1440 minutes. The mean values of MPV and PCT were significantly increased (p<0.00001) along the storage time in both groups. The mean values of PLT and PDW were practically stable (p>0.05) throughout the storage time in both groups.

Avoiding leukocyte contamination and early platelet activation in platelet-rich plasma.

The objective of this study was to describe a new platelet-rich plasma (PRP) protocol with a reduced concentration of leukocytes and intact platelets. We collected 8 mL of venous blood (VB) from marginal ear veins of 10 male New Zealand white rabbits in acid dextrose citrate Vacutainer tubes. Tubes were centrifuged at 302g for 10 minutes. All plasma was collected in plastic tubes to avoid buffy-coat contamination and centrifuged at 2862g for 5 minutes. A 10% calcium chloride activator (10 PRP:2 CaCl2) was added to the lower third of this plasma (PRP), and the PRP gel was obtained. Mean platelet count was 317.7 x 10(3) +/- 39.9/microL in VB and 1344.9 x 10(3) +/- 347.5/microL in PRP. Leukocyte counts were 3.96 x 10(3) +/- 2.01/microL and 0.46 x 10(3) +/- 0.45/microL in VB and PRP, respectively. Mean platelet enrichment was 327.4 +/- 97.8%. All differences were statistically significant (P > .05). This protocol is practical and reproducible, resulting in a high concentration of intact platelets to help tissue repair and low levels of leukocytes.

Effectiveness of two methods for preparation of autologous platelet-rich plasma: an experimental study in rabbits

The purpose of this study was to compare the quantity and quality of platelets in platelet-rich plasma (PRP) samples prepared using either the single- or the double-centrifugation protocol. Ten adult white New Zealand rabbits were used. Ten ml of blood were drawn from each animal via cardiac puncture. Each blood sample was divided into two equal parts for PRP preparation: 5 ml of blood were centrifuged according to a single-centrifugation protocol (Group I), and 5 ml were centrifuged according to a double-centrifugation protocol (Group II). Manual platelet counts were performed on the whole blood and PRP samples of each group. Smears were also done on all samples in order to see the morphology of the platelets. The data obtained in the manual platelet count were submitted to statistical analysis (repeated measures ANOVA, Tukey, P<.05). The average whole blood platelet count was 446,389/μl. The PRP samples in Group II presented an average platelet amount significantly higher than that of Group I (1,986,875 ± 685,020/μl and 781,875 ± 217,693/μl, respectively). The PRP smears from Group II were the only one to present platelets with altered morphology (75% of the smears). A few lymphocytes with increased cytoplasm were observed in the PRP smears of both Groups I (25% of the smears) and II (62.5% of the smears). Within the limits of this study, it can be concluded that the double-centrifugation protocol resulted in higher platelet concentrations than did the single-centrifugation protocol. However, the double-centrifugation protocol caused alterations in platelet morphology and was more sensitive to small processing errors.

Thrombocytopenia and platelet activity in dogs experimentally infected with Rangelia vitalii

The aim of this study was to evaluate the platelet count, coagulation time and platelet activity in dogs experimentally infected with Rangelia vitalii during the acute phase of the disease. For this study, 12 young dogs (females) were used, separated in two groups. Group A (uninfected control) was composed by healthy dogs (n=5), and group B consisted of R. vitalii-infected animals (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite five days post-inoculation (PI). Blood samples were collected on days 0, 10, 20 and 30 PI. The material collected was placed in tubes containing EDTA for quantification of platelets, citrate anticoagulant platelet aggregation, and measuring the clotting time. Right after blood collection on days 10 and 20 PI, dogs were anesthetized for collecting bone marrow samples. A significant reduction (P<0.01) of the number of platelets was observed in R. vitalii-infected blood, when compared with uninfected dogs on days 10 and 20 PI. Additionally, macro-platelets were observed only in infected dogs. Prothrombin time and activated partial thromboplastin time did not differ between infected and uninfected dogs. The megakaryocyte count increased (P<0.01) significantly in infected dogs when compared with uninfected ones on days 10 and 20 PI. Platelet aggregation decreased (P<0.01) significantly in infected dogs in comparison to the control on days 10 and 20 PI. Therefore, rangeliosis in dogs causes a severe thrombocytopenia during the acute phase of infection. This platelets reduction probably occurred due to splenic sequestration and/or immune-mediated thrombocytopenia. (C) 2011 Elsevier B.V. All rights reserved.

Porcine extrahepatic vascular endothelial asialoglycoprotein receptor 1 mediates xenogeneic platelet phagocytosis in vitro and in human-to-pig ex vivo xenoperfusion.

BACKGROUND Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic, femoral arterial, and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS Porcine forelimbs were perfused with whole, heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver, as well as PAEC/PFAEC/PLSEC, were characterized for ASGR1 expression. In vitro, PAEC cultured on microcarrier beads and incubated with non-anticoagulated human blood were used to study binding of human platelets and platelet-white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester-labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1-mediated phagocytosis. RESULTS Human platelet numbers decreased from 102 ± 33 at beginning to 13 ± 6 × 10/μL (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 10/μL). The PAEC, PFAEC, and PLSEC all showed similar ASGR1 expression. In vitro, no correlation was found between reduction in platelet count and platelet-white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester-labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti-ASGR1 antibody and asialofetuin. CONCLUSIONS The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore, ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig-to-primate xenotransplantation.

GPIIIa-(49–66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia

High-affinity (Kd = 1 × 10−9 M) anti-platelet GPIIIa has been isolated from serum immune complexes of immunologic thrombocytopenic HIV-1-infected patients (HIV-1-ITP). Affinity-purified anti-platelet antibody reacted with a recombinant GPIIIa-(1–200) and -(1–66) fusion peptide and with an 18-mer GPIIIa-(49–66) peptide but not with seven other GPIIIa peptides spanning the length of GPIIIa. Most of the anti-platelet antibody (≈85%) could be adsorbed to and eluted from a GPIIIa-(49–66) affinity column. Binding of antibody to platelets could be inhibited by GPIIIa-(49–66) or an equimolar peptide-albumin conjugate (IC50 = 2 μM). Sera from 7 control subjects and 10 classic autoimmune thrombocytopenic patients gave background reactivity with GPIIIa-(49–66). HIV-1-ITP sera from 16 patients reacted with a mean OD 6-fold greater than background (range, 4- to 9-fold). Serum anti-GPIIIa-(49–66) concentration correlated inversely with platelet count, R2 = 0.51, n = 31, P < 0.0001. Because mouse platelet GPIIIa-(49–66) has 83% homology with human GPIIIa and mouse monocytes contain Fc receptors for the human IgG1-κ/λ antibody, we determined the in vivo effect of human anti-GPIIIa on mouse platelets. Affinity-purified antibody, 25–50 μg given i.p., resulted in a precipitous drop in platelet count to 30% of baseline, with nadir at 4 hr and return to normal in 36 hr. No effect was noted with control IgG. Acute thrombocytopenia could be prevented or reversed by the injection of the GPIIIa-(49–66) albumin conjugate at zero time or 2 hr after antibody, respectively, but not with a scrambled peptide-albumin conjugate. Thus HIV-1-ITP patients have high-affinity anti-platelet GPIIIa against a major antigenic determinant, GPIIIa-(49–66), which correlates inversely with platelet count and induces thrombocytopenia in mice.

Angiogenesis is an independent prognostic factor in malignant mesothelioma

Angiogenesis is essential for tumour growth beyond 1 to 2 mm in diameter. The clinical relevance of angiogenesis, as assessed by microvessel density (MVD), is unclear in malignant mesothelioma (MM). Immunohistochemistry was performed on 104 archival, paraffin-embedded, surgically resected MM samples with an anti-CD34 monoclonal antibody, using the Streptavidin-biotin complex immunoperoxidase technique. 93 cases were suitable for microvessel quantification. MVD was obtained from 3 intratumoural hotspots, using a Chalkley eyepiece graticule at × 250 power. MVD was correlated with survival by Kaplan-Meier and log-rank analysis. A stepwise, multivariate Cox model was used to compare MVD with known prognostic factors and the EORTC and CALGB prognostic scoring systems. Overall median survival from the date of diagnosis was 5.0 months. Increasing MVD was a poor prognostic factor in univariate analysis (P = 0.02). Independent indicators of poor prognosis in multivariate analysis were non-epithelial cell type (P = 0.002), performance status > 0 (P = 0.003) and increasing MVD (P = 0.01). In multivariate Cox analysis, MVD contributed independently to the EORTC (P = 0.006), but not to the CALGB (P = 0.1), prognostic groups. Angiogenesis, as assessed by MVD, is a poor prognostic factor in MM, independent of other clinicopathological variables and the EORTC prognostic scoring system. Further work is required to assess the prognostic importance of angiogenic regulatory factors in this disease. © 2001 Cancer Research Campaign.

Tumour necrosis is an independent prognostic marker in non-small cell lung cancer : correlation with biological variables

Background Tumour necrosis (TN) is recognized to be a consequence of chronic cellular hypoxia. TN and hypoxia correlate with poor prognosis in solid tumours. Methods In a retrospective study the prognostic implications of the extent of TN was evaluated in non-small cell lung cancer (NSCLC) and correlated with clinicopathological variables and expression of epidermal growth factor receptor, Bcl-2, p53 and matrix metalloproteinase-9 (MMP-9). Tissue specimens from 178 surgically resected cases of stage I-IIIA NSCLC with curative intent were studied. The specimens were routinely processed, formalin-fixed and paraffin-embedded. TN was graded as extensive or either limited or absent by two independent observers; disagreements were resolved using a double-headed microscope. The degree of reproducibility was estimated by re-interpreting 40 randomly selected cases after a 4 month interval. Results Reproducibility was attained in 36/40 cases, Kappa score=0.8 P<0.001. TN correlated with T-stage (P=0.001), platelet count (P=0.004) and p53 expression (P=0.031). Near significant associations of TN with N-stage (P=0.063) and MMP-9 expression (P=0.058) were seen. No association was found with angiogenesis (P=0.98). On univariate (P=0.0016) and multivariate analysis (P=0.023) TN was prognostic. Conclusion These results indicate that extensive TN reflects an aggressive tumour phenotype in NSCLC and may improve the predictive power of the TMN staging system. The lack of association between TN and angiogenesis may be important although these variables were not evaluated on serial sections. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer

Purpose To evaluate carbonic anhydrase (CA) IX as a surrogate marker of hypoxia and investigate the prognostic significance of different patterns of expression in non-small-cell lung cancer (NSCLC). Methods Standard immunohistochemical techniques were used to study CA IX expression in 175 resected NSCLC tumors. CA IX expression was determined by Western blotting in A549 cell lines grown under normoxic and hypoxic conditions. Measurements from microvessels to CA IX positivity were obtained. Results CA IX immunostaining was detected in 81.8% of patients. Membranous (m) (P = .005), cytoplasmic (c) (P = .018), and stromal (P < .001) CA IX expression correlated with the extent of tumor necrosis (TN). The mean distance from vascular endothelium to the start of tumor cell positivity was 90 μm, which equates to an oxygen pressure of 5.77 mmHg. The distance to blood vessels from individual tumor cells or tumor cell clusters was greater if they expressed mCA IX than if they did not (P < .001). Hypoxic exposure of A549 cells for 16 hours enhanced CAIX expression in the nuclear and cytosolic extracts. Perinuclear (p) CA IX (P = .035) was associated with a poor prognosis. In multivariate analysis, pCA IX (P = .004), stage (P = .001), platelet count (P = .011), sex (P = .027), and TN (P = .035) were independent poor prognostic factors. Conclusion These results add weight to the contention that mCA IX is a marker of tumor cell hypoxia. The absence of CA IX staining close to microvessels suggests that these vessels are functionally active. pCA IX expression is representative of an aggressive phenotype. © 2003 by American Society of Clinical Oncology.

The acute effects of eccentrically-biased versus conventional weight training in older adults : a randomised controlled cross-over study

Background Whilst resistance training has been proven to convey considerable benefits to older people; immediately post-exercise there may be elevated transient risks for cardiac events and falls. Objectives and Measurements We assessed the acute effects of eccentrically-biased (EB) and conventional (CONV) resistance exercise on: platelet number, activation and granule exocytsosis; and mean velocity of centre of pressure displacement (Vm). Design, Setting, Participants and Intervention Ten older adults (7 males, 3 females; 69 ± 4 years) participated in this randomised controlled cross-over study in which they performed EB and CONV training sessions that were matched for total work and a control condition. Results Immediately post-exercise there was a statistically significant difference in platelet count between the control condition, in which it fell (pre 224 ± 35 109/L; post 211 ± 30 109/L: P < 0.05) and CONV in which it increased (pre 236 ± 55 109/L; post 242 ± 51 109/L: P > 0.05). There was no change in platelet activation and granule exocytsosis or Vm following EB and CONV. Conclusions Overall, while minor differences between regimens were observed, no major adverse effect on parameters of platelet function or centre of pressure displacement were observed acutely following either regimen. Eccentrically-biased and conventional resistance exercise training regimens do not appear to present an elevated acute risk in the context of changes to platelet function contributing to a cardiac event or postural stability increasing falls risk for apparently healthy older adults.