Platelet activation responses in vitro to human mast cell activation

Mast cells are thought to play an important role in atherogenesis and plaque rupture, but their role in the subsequent platelet activation and thrombus formation is unclear. Tryptase positive cells (KU812T+) were established from the KU812 cell line as an in vitro model of human mast cells and used to study the effect of mast cell activation on human platelets. Overnight incubation of KU812T+ with IgE and subsequent challenge with anti-IgE caused the release of heparinoid substances which inhibited 1 microg/ml collagen-induced platelet aggregation. KU812T+ challenged with compound 48/80 produced a releasate that had no apparent heparinoid content but caused full platelet aggregation. These findings showed that, although activation of KU812T+ via FcepsilonR1 partially abrogated collagen-induced platelet aggregation, activation of the C5a receptor signalling pathway, by compound 48/80, caused the release of potent platelet-activating substances. This cell culture model offers a unique insight into the role of platelet-mast cell interactions in arterial thrombogenesis.     

Platelet count and platelet size in males and females

The platelet count of healthy males was compared to that of healthy females. A higher platelet count in women was confirmed. A similar inverse non-linear relationship between the platelet count and the mean platelet volume (MPV) was found in the two sexes. The slightly higher MPV in men was insufficient to compensate for the lower platelet count so that the plateletcrit, which measures total volume of platelets in a given volume of blood, was also significantly higher in women.     

Platelet activation in Helicobacter pylori-associated idiopathic thrombocytopenic purpura: eradication reduces platelet activation but seldom improves platelet counts

INTRODUCTION: It has been suggested that Helicobacter pylori eradication often increases platelet counts in patients with chronic idiopathic thrombocytopenic purpura (ITP). In addition, H. pylori has been shown to induce platelet activation (CD62p or P-selectin expression) in previous studies. We assessed the response of platelet count and CD62p expression after eradication therapy in patients with ITP and H. pylori infection. METHODS AND RESULTS: We prospectively studied 15 ITP patients diagnosed with H. pylori infection by serology and breath test. A follow-up breath test was used to document eradication. Two out of 15 patients showed improvement in platelet counts after 6 months, 1 of which may have had drug-induced thrombocytopenia. Overall, certain platelet response rate in our series was 6.7% (1/15). We found that platelet CD62p expression by flow cytometry was elevated in 10/15 (66.7%) H. pylori-infected patients, which is a statistically significant difference when compared with 3/33 (9.1%) control ITP patients seronegative for H. pylori (p = 0.002). In addition, eradication therapy decreased CD62p expression (p = 0.04). However, reduction in platelet activation was not associated with an increase in platelet counts (mean 72.4 x 10(9)/l before and 68.7 after therapy; p = 0.4). CONCLUSION: In our series, platelet activation was common in ITP patients with H. pylori, and eradication therapy decreased platelet activation but seldom increased platelet counts. Increased platelet CD62p expression is a putative link between chronic infections and atherosclerosis, but further study is needed to clarify the implications of our observation.     

Platelet size does not correlate with platelet age

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense- body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.     

Platelet size and age determine platelet function independently

This study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se- methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms.     

Platelet activation induces metalloproteinase-dependent GP VI cleavage to down-regulate platelet reactivity to collagen

Glycoprotein (GP) VI, the primary collagen receptor on platelets, has been shown to have variable expression, possibly as a consequence of immune modulation. The present study was designed to determine the mechanism by which GP VI clearance occurs. We found that direct activation of GP VI both by a GP VI-specific antibody and by GP VI ligands (collagen and convulxin) reduced binding of biotinylated convulxin to the stimulated platelets. Analysis of immunoblots of platelets and supernatants showed that the stimulated platelets contained less GP VI, while the soluble fraction contained a 57-kDa cleavage product. Stimulation of platelets with PAR-1 agonists (TRAP peptide and thrombin) also caused GP VI cleavage, although the amount of GP VI loss was less than that observed with direct GP VI ligands. The metalloproteinase (MMP) inhibitors GM6001 and TAPI prevented both the clearance of GP VI from the platelet surface and the appearance of the soluble cleavage product. Induction of GP VI cleavage caused specific down-regulation of collagen-induced platelet aggregation, providing a mechanism for the modulation of platelet responsiveness to this important platelet agonist.     

Platelet count and platelet size in healthy Africans and West Indians

Healthy subjects of African origin were found to have lower platelet counts than simultaneously studied healthy Caucasians. A similar reduction was found in a large group of West Indian females, though not in a small group of West Indian males. It is suggested that the lower platelet counts observed in Africans and West Indians may be partly environmental and partly genetic in origin. The mean platelet volume and platelet distribution width in the Africans and West Indians showed the same relationship with the platelet count as was found in Caucasians, so that the same nomograms can be used in all ethnic groups to assess whether there is deviation from normality.     

Platelet function measured by VerifyNow identifies generalized high platelet reactivity in aspirin treated patients

Selected aspirin treated patients may exhibit high platelet reactivity to agonists other than arachidonic acid. This study aimed to determine whether the VerifyNow identifies generalized high platelet reactivity supported by correlations with other established methods that stimulate platelets with various agonists. Stable outpatients with coronary artery disease (n = 110) were treated with aspirin in a two 3 x 3 Latin square design (81, 162 and 325 mg/day for 4 weeks each). VerifyNow (arachidonic acid (AA) cartridge); light transmittance aggregometry; thrombelastography; PFA-100; flow cytometry; PlateletWorks; and urinary 11- dehydro thromboxane levels were measured. Multianalyte profiling measured fibrinogen and von Willebrand factor (vWF). Patients with >or=550 ARU by VerifyNow had increased 5 mM AA-, 5 microM ADP-, and 2 microg/mL collagen-induced platelet aggregation compared to patients with <550 ARU (p 相似文献   

Platelet genomics: the role of platelet size and number in health and disease

Taken together, there is ample evidence of the association of cardiovascular disease, cerebrovascular, and inflammatory disease with single nucleotide variants (SNV) due to their impact on platelet size, number, and function. With the use of electronic medical record (EMR) or other phenotypic-linked bioinformatics sources, the more important “functional” variants are emerging and provide valuable information on their specific role in promoting early onset of disease or poor response to therapeutic measures. This review will focus upon the recognized common polymorphisms or gene variants with small, but functional effects, as it is becoming clear that these contribute to hyper- or hypo-responsive platelet phenotypes. The impact of these gene variants is distinguishable among normal individuals, and they are suspected contributors to increased risk of adverse outcomes in patients with underlying disease. There are thousands of gene variants and environmental factors that may mitigate risk or amplify the potential for disease within each of us. When combined with the environment and epigenetic influences, it is clear that whole-genome sequencing and bioinformatics alone will not be enough to truly predict “risk” or probability, but awareness of their potential influence may be a starting point in selective screening and generating prevention strategies to promote a healthy lifestyle or fine-tune therapeutic choices in the future.     

Low-dose aspirin does not lower in vivo platelet activation in healthy smokers

Smoking causes atherosclerosis, and smokers have increased thromboxane (TXA₂) formation. As aspirin inhibits TXA₂ production we speculated that smokers would preferentially profit from inhibition of the TXA₂ pathway by aspirin. Increased expression of P-selectin, a constituent of the alpha-granules of platelets, and increased levels of circulating (c)P-selectin in plasma are markers for platelet activation. The aim of this study was to compare P-selectin expression on platelets between smokers and nonsmokers, and to compare with placebo the effect of 2 weeks administration of 100 mg/d aspirin on platelet activation in smokers. Smokers exhibited higher P-selectin expression on platelets than non-smokers (2.7 ± 1.8% v 1.6 ± 0.6%, P = 0.018), thus confirming increased platelet activation. Aspirin did not reduce platelet activation as demonstrated by unchanged P-selectin expression on platelets and cP-selectin plasma levels.     

Platelet contribution to the formation of metastatic foci: the role of cancer cell-induced platelet activation

Platelets are thought to be involved in the development of blood borne metastasis. Ultrastructural and experimental studies demonstrate that association between tumor cells and platelets with subsequent activation of the coagulation cascade takes place in malignancy. Several hypotheses have been proposed to explain the mechanisms by which tumor cells activate platelets including generation of thrombin, ADP release and involvement of arachidonate metabolism. Perfusion studies with human homologous systems showed that intact tumor cells and tumor cell microvesicles were able to induce platelet thrombogenicity under defined flow conditions. The presence of divalent cations and plasma factors was necessary for the cancer cells to exert their activating capacity. These results suggest a role for platelets in the development of secondary metastasis as well as in the thrombotic events of malignancy.     

Platelet activation is increased in patients with cardiomyopathy: myocardial inflammation and platelet reactivity

Thrombotic events are a major complication in patients with cardiomyopathy, in which inflammation is often found within the heart. We examined the platelet activation in patients with cardiomyopathy with and without myocardial infiltrates. Endomyocardial biopsies of 45 patients with cardiomyopathy (CM) were immunohistologically assessed for infiltrates. Twenty-three patients had myocardial infiltrates ( S 2 CD3 + cells/high power field (HPF), CM+) and 22 patients had no inflammation (< 2 CD3 + cells/HPF, CM-). Platelet adhesion proteins were flow cytometrically quantified (thrombospondin, P-selectin, CD 41) and platelet activation in CM compared to 45 healthy controls. Significantly more activated platelets were detected in patients with cardiomyopathy than controls (for thrombospondin 13.5% [10.3; 22.0] median [25; 75 quartile] vs. 10.6% [8.2; 16.0], P = 0.002; for P-selectin 12.6% [10.0; 18.6] vs. 7.7% [5.8; 10.9], P < 0.001). Platelet activation was higher in patients with cardiomyopathy and myocardial infiltrates (for thrombospondin 19.0% [11.0; 26.3]) compared to patients without inflammation (12.3% [9.9; 16.0], P = 0.018). Platelet GPIIb/IIIa expression was also increased in patients with inflammation (290 arbitrary units [268, 338]) compared to the controls (215 [188, 248], P < 0.001). In conclusion, platelet reactivity was increased in patients with cardiomyopathy and myocardial infiltrates. Measurement of platelet reactivity may be useful to identify patients with cardiomyopathy at risk for thrombotic events.     

Platelet activation is increased in patients with cardiomyopathy: myocardial inflammation and platelet reactivity

Thrombotic events are a major complication in patients with cardiomyopathy, in which inflammation is often found within the heart. We examined the platelet activation in patients with cardiomyopathy with and without myocardial infiltrates. Endomyocardial biopsies of 45 patients with cardiomyopathy (CM) were immunohistologically assessed for infiltrates. Twenty-three patients had myocardial infiltrates (>/= 2 CD3(+) cells/high power field (HPF), CM+) and 22 patients had no inflammation (< 2 CD3(+) cells/HPF, CM-). Platelet adhesion proteins were flow cytometrically quantified (thrombospondin, P-selectin, CD 41) and platelet activation in CM compared to 45 healthy controls. Significantly more activated platelets were detected in patients with cardiomyopathy than controls (for thrombospondin 13.5% [10.3; 22.0] median [25; 75 quartile] vs. 10.6% [8.2; 16.0], P = 0.002; for P-selectin 12.6% [10.0; 18.6] vs. 7.7% [5.8; 10.9], P < 0.001). Platelet activation was higher in patients with cardiomyopathy and myocardial infiltrates (for thrombospondin 19.0% [11.0; 26.3]) compared to patients without inflammation (12.3% [9.9; 16.0], P = 0.018). Platelet GPIIb/IIIa expression was also increased in patients with inflammation (290 arbitrary units [268, 338]) compared to the controls (215 [188, 248], P < 0.001). In conclusion, platelet reactivity was increased in patients with cardiomyopathy and myocardial infiltrates. Measurement of platelet reactivity may be useful to identify patients with cardiomyopathy at risk for thrombotic events.     

Platelet size analysis in the quality assurance of platelet concentrates for transfusion

Summary The platelet distribution width (PDW) as analysed on standard haematology cell counters is an indicator of size dispersion in the platelet population. Using a Sysmex® E-2500 analyser platelet concentrates prepared for transfusion showed an increase in PDW over storage. This increase correlated strongly with in vitro indicators of platelet viability (pH and response to osmotic stress). PDW may thus be useful for clinical haematology laboratories as a predictor of the viability of transfused platelets. The same instrument gave a measure of the largest platelets in the platelet population as a large cell ratio (P-LCR). For platelet concentrates with less than 8 × 10¹⁰ platelets/unit, the P-LCR at preparation was negatively associated with the end of storage pH, indicating that the presence of large platelets increases the production of lactic acid and accelerates the platelets’ metabolic storage lesion. This information may be useful in determining storage conditions for single donor platelets harvested by apheresis.     

Deposition of insect cuticle in vitro: differential responses to alpha- and beta-ecdysone

A series of time-dose studies was made to study the effects of α- and β-ecdysone on the induction of cuticle deposition by leg regenerates of the cockroach Leucophaea maderae (F.). With β-ecdysone, similar contributions to the frequency of cuticle deposition were made by the concentration of and length of exposure to the hormone. The ability of α-ecdysone to induce cuticle deposition was time-dependent. Thus, the action of α-ecdysone required an additional rate-limited process that was not required for the action of β-ecdysone. Subsequent experiments showed that the action of α-ecdysone was inhibited by the presence of puromycin but that that of β-ecdysone was not. These findings indicate that the activity of α-ecdysone in inducing cuticle deposition in vitro is dependent on its enzymatic conversion to β-ecdysone. An overview of the literature suggests that these processes differ in tissues of insect from different orders.