Alpha-granule Membrane Research Articles

PIKfyve-Dependent Phosphoinositide Dynamics in Megakaryocyte/Platelet Granule Integrity and Platelet Functions.

Secretory granules are key elements for platelet functions. Their biogenesis and integrity are regulated by fine-tuned mechanisms that need to be fully characterized. Here, we investigated the role of the phosphoinositide 5-kinase PIKfyve and its lipid products, PtdIns5P (phosphatidylinositol 5 monophosphate) and PtdIns(3,5)P2 (phosphatidylinositol (3,5) bisphosphate) in granule homeostasis in megakaryocytes and platelets. For that, we invalidated PIKfyve by pharmacological inhibition or gene silencing in megakaryocytic cell models (human MEG-01 cell line, human imMKCLs, mouse primary megakaryocytes) and in human platelets. We unveiled that PIKfyve expression and its lipid product levels increased with megakaryocytic maturation. In megakaryocytes, PtdIns5P and PtdIns(3,5)P2 were found in alpha and dense granule membranes with higher levels in dense granules. Pharmacological inhibition or knock-down of PIKfyve in megakaryocytes decreased PtdIns5P and PtdIns(3,5)P2 synthesis and induced a vacuolar phenotype with a loss of alpha and dense granule identity. Permeant PtdIns5P and PtdIns(3,5)P2 and the cation channel TRPML (transient receptor potential mucolipin) 1 and TPC (two pore segment channel) 2 activation were able to accelerate alpha and dense granule integrity recovery following release of PIKfyve pharmacological inhibition. In platelets, PIKfyve inhibition specifically impaired the integrity of dense granules culminating in defects in their secretion, platelet aggregation, and thrombus formation. These data demonstrated that PIKfyve and its lipid products PtdIns5P and PtdIns(3,5)P2 control granule integrity both in megakaryocytes and platelets.

Zn(II) coordination polymers: therapeutic activity and nursing application values against coronary heart disease

ABSTRACT In the current research, two coordination polymers (CPs) have been produced solvothermally on the basis of a semi-rigid multifunctional tricarboxylate, i.e., 5-(3,4-dicarboxylphenoxy) nicotic acid (H3L), and the chemical compositions of the two compounds are [Zn(H2L)2(H2O)2] 1 and [Zn(HL)(2,2'-bpy)] (2, 2,2'-bpy = 2,2'-bipyridine), respectively. The structures and CHN analysis of both complexes were researched. The structural analysis results show that complex 1 features a 2D layered network with sql-type topology and complex 2 demonstrates a 2D layered network with uninodal hcb topology. The therapeutic activity and nursing application values of compounds against coronary heart disease were explored, and their relevant mechanism was assessed in meantime. The endothelin (ET) and prostacyclin (PGI2) contents released by the arterial endothelial cells into plasma were determined with ELISA assay. In addition to this, the alpha granule membrane protein 140 (GMP140) on the platelet was determined with real-time RT-PCR assay.

Correlation and mechanism between cardiac magnetic resonance imaging and oral streptococcus count in patients with primary microvascular angina pectoris.

Although primary microvascular angina (PMVA) can be diagnosed clinically, the etiology and pathophysiology of PMVA remain unclear. The effects of conventional clinical medications (aspirin, statins, and nitrates) are unsatisfactory, and PMVA can lead to serious cardiovascular events. The present study was designed to analyze the correlation between the load perfusion cardiovascular magnetic resonance imaging (CMR) results and the Streptococcus sanguinis(S sanguinis) count and the correlations between the S sanguinis count in oral cavity subgingival plaque and changes in the plasma levels of platelet alpha-granule membrane glycoprotein 140 (GMP-140), fibrinopeptide A (FPA), von Willebrand factor (vWF), and homocysteine (Hcy) in patients with PMVA after increased anti-infective treatment of the oral cavity. This study also discusses the pathogenesis of PMVA from this perspective. The differences in the S sanguinis count in oral cavity subgingival plaque and oral health status between healthy people and PMVA patients will be compared, and the correlation between the oral cavity health status and disease in PMVA patients will be analyzed. The present randomized controlled trial with a parallel control group will be conducted in 68 PMVA patients diagnosed by the in-patient cardiology department. The selected patients will be randomly divided into 2 groups, one receiving routine drug treatment and the other a combination of anti-infective treatments. The normal control group will comprise 30 healthy people with no infectious oral cavity disease matched by age and sex. We will conduct CMR, and the presence of S sanguinis in subgingival plaques will be used to determine the bacterial count in PMVA patients. Blood samples will also be collected to determine the levels of GMP-140, FPA, vWF, and Hcy. S sanguinis in the subgingival plaque of PMVA patients will be further analyzed after increasing the oral cavity anti-infective treatment; the resulting changes and their correlations with changes in GMP-140, FPA, vWF, and Hcy levels will be assessed. Additionally, the differences in the S sanguinis count and the oral cavity health status of oral cavity dental plaque between healthy people and PMVA patients will be determined, and the correlation between the oral cavity conditions and PMVA will be analyzed. The relationship between the perfusion CMR results and the oral cavity S sanguinis count of PMVA patients, and the potential pathogenesis, will be explored. We will use the SPSS19.0 statistical software package to analyze the data. The measurements will be expressed as means±standard deviation. Student t test will be used for intergroup comparisons, a relative number description will be used for the count data, and the chi-square test will be used for intergroup comparisons. Multivariate logistic regression will be performed to identify associations. A P value < .05 will be considered significant. In this study, the correlation between the perfusion CMR results and the S sanguinis count in oral cavity subgingival plaque of PMVA patients will be analyzed. Changes in the levels of GMP-140, FPA, vWF, and Hcy of PMVA patients after receiving increased oral cavity anti-infective treatment will be explored, and the difference in the S sanguinis count in oral cavity subgingival plaque and the oral cavity health status between healthy people and PMVA patients will be compared. Chinese Clinical Trial Registry, (http://www.chictr.org.cn/showprojen.aspx?proj=45091).

Platelet Membrane Procoagulation in Preeclampsia

Background: Preeclampsia is a hypertensive disorder in pregnancy that results in significant adverse maternal and neonatal outcomes. Platelet activation is involved in the pathophysiology of preeclampsia and contributes to the prothrombotic state of the disorder. The mechanisms which initiate and sustain platelet activation in preeclampsia, and how platelets are involved in the pathogenesis of coagulation abnormalities in preeclampsia remains unclear.Aim: To utilise a systematic analysis of Procoagulant Membrane Dynamics (PMD) to assess platelet procoagulation in normotensive pregnancies and preeclampsia, compared to age-matched non-pregnant female controls. Methods: Platelets were assessed using 4D live-cell imaging, immunoassay, flow cytometry and impedance aggregometry. Study participants were healthy, non-diabetic, normotensive, non-pregnant controls, (NP, N=9), normotensive pregnant controls (PC, N=9), and pregnant women with preeclampsia (PEE, N=8). Result: Platelets of preeclamptic patients were significantly activated under basal conditions and showed major remodelling of the open canalicular or dense tubular system. Compared to NP and PC controls, PEE platelets showed a marked decrease in dense granule release, and total exposed phosphatidylserine (PS)-laden surface as well as a decreased whole-blood aggregation in response to high concentration collagen stimulation. Notably, in patients who were pregnant, we visualized in both plasma and peripheral whole blood, freely suspended platelet microthrombi ranging from 10-15 µm radius but were more in PEE participants (~75%) than in the normal pregnant controls (~22%). These microthrombi showed fibrin deposits in between procoagulant platelets with ballooned membranes. Unstimulated PEE platelets showed more than a 2-fold increase in the membrane expression of the facultative glucose transporter GLUT3 which normally reside within the intracellular membrane of alpha granules. Multiplex immunoassay for inflammatory markers confirmed the elevation of previously unreported i-309 and C-TACK inflammatory cytokines in PEE participants. Discussion: We elucidated a platelet-based mechanism of procoagulation in patients with preeclampsia; and demonstrated that platelets were highly activated in the basal, resting state in preeclampsia. Platelet hyperactivation in preeclampsia is complex; there is evidence of procoagulant morphologic membrane changes and p-selectin expression, yet platelets were less able to adhere to exposed collagen to form primary hemostatic plugs. Additionally, novel platelet activation and inflammatory markers such as GLUT3, i-309, and C-TACK cytokines requires further study to understand how they mediate the platelet activation process and the pathophysiology of preeclampsia. Conclusion: Platelet hyperactivation and increased GLUT3 membrane expression may contribute to the progression of preeclampsia, and to the thrombotic and coagulopathic maternal complications of the disease. The platelet activation process itself may provide biomarkers for staging and phenotyping preeclampsia and for the prediction of its severity, clinical manifestations, and disease progression. Disclosures Lee: Takeda: Consultancy, Honoraria, Speakers Bureau; Bayer: Honoraria. Poon:Takeda: Other: honoraria for advisory board meeting attendance; Pfizer: Other: honoraria for advisory board meeting attendance; Novo Nordisk: Other: honoraria for advisory board meeting attendance; Roche: Other: honoraria for advisory board meeting attendance; Bayer: Other: honoraria for advisory board meeting attendance, Research Funding; Bioverative/Sanofi: Other: honoraria for advisory board meeting attendance; CSL-Behring: Other: honoraria for advisory board meeting attendance, Research Funding.

Evidence of platelet dysfunction in congenital cyanotic heart disease: relevance to palliative systemic-pulmonary shunt and haemostatic parameters

Abstract Introduction Platelets in congenital cyanotic heart disease (CCHD) were occasionally characterized by significantly enhanced expression of P-selectin in resting circulating platelets as well as their augmented activation in response to stimulating agents. P-selectin is present in the alpha granule membrane as well as Weibel-Palade body membrane of endothelial cells. It is translocated to platelet membrane after activation; thus, its expression on platelet surface is an indicator of platelet activation in vivo. The occurrence of the prothrombotic variant of GPIIIa in cyanotic children may be associated with enhanced activation of circulating platelets. Purpose To study the platelet functions in patients with CCHD by determination of Pselectin expression and the GPIIIa polymorphism and to correlate these findings with other clinical, radiological, and laboratory parameters in these patients. Methods This study included 47 patients, with cyanotic congenital heart disease with decreased pulmonary blood flow, attending the Pediatric Cardiology Clinic. They were divided into two groups: Group I: 31 patients with congenital cyanotic heart disease before cardio surgery Group II: 16 patients with congenital cyanotic heart disease 3–6 months after palliative or corrective cardio surgery, Group III: 12 age and sex matched normal children were included in the study as control group. They were subjected to Flow-cytometric evaluation of platelet activation by Pselectin expression: P-selectin expression was estimated using monoclonal antibody.-Typing of GPIIIa gene polymorphism was done. Results Statistically significant higher P-selectin levels were found in patients compared with controls being significantly higher in patients &amp;lt;3 years of age. There was significant negative correlation between P-selectin level and O2 in patients. The incidence of prothrombotic variant A2 was described as phenotype positive. Statistically significant higher levels of P-selectine were found in positive phenotype patients than positive phenotype controls Conclusion Enhanced platelet activation may be an important contributor in the high thrombotic liability in congenital cyanotic heart disease. This may be attributed in part to genetic factors Funding Acknowledgement Type of funding source: None

Effects of remote ischemic post-conditioning on platelet activation of AMI patients.

The recovery of ischemic myocardium blood perfusion is the main treatment option for acute myocardial infarction (AMI). However, this treatment option has multiple side effects that directly affect the quality of life of the patients. The activation of platelet function plays an important role in the occurrence, development and treatment of AMI. The aim of the present study was to analyze the effects of remote ischemic post-conditioning on platelet activation of AMI patients with primary PCI treatment and clinical prognosis. A total of 71 patients with AMI were treated with primary percutaneous coronary intervention (PCI). They were randomly divided into control group (n=34) and observation group (n=37). The patients in the observation group were treated with remote ischemic post-conditioning. Further, flow cytometer was used to detect the platelet alpha granule membrane glycoprotein (CD62P) and the percentages of activated IIb/IIIa (PAC-1). The maximum platelet aggregation rate induced by adenosine diphosphate (ADP) and arachidonic acid (AA) was measured by light transmittance aggrometer. The incidence of major adverse cardiac events (MACE) was compared between the two groups during the follow-up period of 6 months. The percentage of CD62P (24 h after PCI) in the observation group was significantly lower than control group (P<0.05). Further, the incidence of MACE in the observation group was also lower than that of the control group (P<0.05). Remote ischemic post-conditioning could reduce the incidence of MACE in patients with AMI after primary PCI treatment. Moreover, the above observation may be related to the improvement of platelet CD62P activation.

Effects of different anticoagulants on coagulation function in hemodialysis patients

Objective To investigate the effect of different anticoagulant drugs on the changes of coagulation index and the incidence of hemorrhage in hemodialysis patients. Methods 40 patients with hemodialysis were selected as study objects, according to the random number table method they were divided into three groups, 20 cases received heparin in A group, 10 cases received low molecular heparin in B group; 10 cases received argatroban in C group.At the same time, 10 healthy persons were selected as control group.The activated glass bead clotting time (gbACT), blood velocity (CR), platelet function (PF), prothrombin fragment 1+ 2 (PF1+ 2) and the surface of platelet alpha granule membrane protein (GMF-140) were observed and analyzed in all patients. Results Compared with the control group, the CR, PF, PF1+ 2 and GMF-140 before taking to machine in A group were significantly increased, which were (31.1±5.5)sig/min, (3.1±0.5), (478.2±74.3)pmol/L, (36.9±6.6)pmol/L, respectively, gbACT was significantly prolonged [(196.1±27.3)s]. In A group, compared with before taking to machine, the CR, PF, PF1+ 2 and the GMF-140 before taking off machine were significantly decreased, which were (10.6±3.2)sig/min, (1.8±0.4), (400.1±85.5)pmol/L, (30.9±6.8)pmol/L, respectively, gbACT was significantly prolonged[(252.5±50.4)s], there were statistically significant differences (F=112.64, 28.38, 81.40, 18.11, 21.63 0.05, all P<0.05); B group compared with the control group, the CR, PF, PF1+ 2 and GMF-140 before taking to machine were significantly increased, which were (29.5±6.6)sig/min, (2.9±1.2), (475.5±97.0)pmol/L, (37.5±7.0)pmol/L, respectively, gbACT was significantly prolonged[(193.9±32.8)s]. In B group, the CR and PF1+ 2 before taking off machine were significantly decreased, which were (22.3±6.8), (407.8±90.3)pmol/L, respectively, gbACT was significantly prolonged [(207.5±31.5)s], there were statistically significant differences (F=9.11, 57.81, 5.99, 16.37, all P<0.05); C group compared with the control group, the CR, PF, PF1+ 2 and GMF-140 before taking to machine were significantly increased, which were (26.1±3.3)sig/min, (2.5±0.5), (443.5±64.1)pmol/L, (32.2±8.3)pmol/L, respectively, before taking off machine, the CR[(23.7±4.1)] was higher than (18.5±3.6) of the control group, there were statistically significant differences (F=11.14, 3.04120.30, 8.63, all P<0.05).40 hemodialysis patients were successfully treated without the occurrence of elevated venous pressure during and after treatment pipeline no or slight residual blood, puncture time and no significant prolongation. Conclusion Unfractionated heparin has strong anti-clotting effect, during and after hemodialysis has greater risk of bleeding, dalteparin has anticoagulant effect, but there is greater risk of bleeding during hemodialysis, the use of argatroban helps to improve the condition of bleeding. Key words: Hemodialysis; Anticoagulant drugs; Heparin; Heparin sodium; Aplus

Abstract 56: Platelet Specific Knockout of Glucose Transporter 3 Leads to Altered Metabolism and Decreased Platelet Activation

Patients with diabetes display increased thrombosis and platelet activation. In these disorders, the systemic milieu is characterized by multiple metabolic changes including increased glucose concentrations. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. Therefore we hypothesized that decreasing platelet glucose uptake would limit glycolysis thereby decreasing energy production and platelet reactivity. Platelets import glucose via two glucose transporters GLUT1 and GLUT3. GLUT1 is expressed on the plasma membrane and GLUT3 is expressed predominantly on alpha granule membranes (85%) and to a lesser extent on the plasma membrane (15%). To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout (KO) of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. Platelet glycogen content and glycolytic intermediates were significantly reduced in GLUT3 KO platelets compared to controls, and following mitochondrial uncoupling exhibited reduced glycolysis rates. Interestingly, under these conditions, mitochondrial maximal respiration was increased two-fold, with no change in mitochondrial density, or citric acid cycle intermediates. In vitro , GLUT3 deficient platelets display a 90% reduction of spreading on fibrinogen and collagen matrixes and significant reductions in CD62p surface translocation and GPIIbIIIa activation following stimulation with multiple agonists. Additionally makers of alpha granule release were significantly reduced. In vivo analysis of GLUT3 KO mice using a 10% ferric chloride model of arterial thrombosis and a tail-bleed model indicated no alteration in thrombosis between littermate controls and knockouts. However in a KBx/N model of rheumatoid arthritis GLUT3 KO mice exhibited significantly reduced disease severity. Together, these data indicate that GLUT3-mediated glucose uptake is essential for platelet activation, spreading and alpha granule release. GLUT3 modulates mechanisms that promote rheumatoid arthritis but not those that regulate in vivo thrombus formation.

P-Selectin Expression and Platelet Function Are Developmentally Regulated

Platelets have numerous well-defined roles during adult hemostasis, however little is known about whether these functions are developmentally regulated. In the adult, P-selectin is constitutively expressed in megakaryocytes and localized to the membrane of alpha granules. Activation of platelets results in granule secretion and P-selectin surface exposure allowing for recruitment of leukocytes to sites of vascular injury and the formation of leukocyte-platelet aggregates. Here, we examined the expression and function of P-selectin in platelets during murine embryogenesis. Platelets were isolated from timed pregnant outbred-ICR mice at embryonic day 12.5 (E12.5), E15.5, postnatal day 1 (PN1), PN7, PN21, and were assayed for thrombin induced integrin activation and P-selectin translocation. While no significant differences were seen in the ability of embryonic platelets to be activated by thrombin, P-selectin surface expression after thrombin stimulation was significantly reduced in embryonic compared to adult platelets (E15.5 p=.0006, E12.5 p=.0004, PN1 p=.01, n=3, unpaired t-test). Surprisingly, P-selectin expression began to appear on the surface of activated platelets only after birth, approaching 50% and 75% of maternal expression at PN7 and PN21, respectively. No significant difference was seen in VEGF secretion after thrombin activation of E12.5, E15.5 and adult platelets (n=5), suggesting that the lack of P-selectin surface expression was not due to a loss of alpha granule secretion. To further characterize expression of P-selectin, real-time qPCR was performed on mRNA harvested from platelets at E12.5, E15.5, PN1 and adult stages. P-selectin transcript levels were significantly lower in embryonic and PN1 platelets, suggesting that P-selectin expression is transcriptionally regulated. Consistent with this concept, Imagestream analysis indicates that P-selectin protein is absent in megakaryocytes derived from the fetal liver. P-selectin expression on platelets is an important mediator of platelet-leukocyte interactions; therefore, we assayed the ability of platelets to bind to leukocytes in embryonic whole blood. Preliminary data suggest that embryonic platelets do not form leukocyte-platelet aggregates upon ex vivo stimulation with AYPGKF, a Par4 agonist. Taken together, our data indicate that P-selectin expression is developmentally regulated in primary mouse platelets. The absence of P-selectin expression in embryonic platelets correlates with an impaired ability to interact with leukocytes. DisclosuresNo relevant conflicts of interest to declare.

Effect of intracoronary tirofiban on platelet alpha-granule membrane protein and myocardial perfusion level during emergency percutaneous coronary intervention.

This study aimed to investigate the effect of intracoronary application of tirofiban on platelet alpha-granule membrane protein (GMP-140) and myocardial perfusion levels during emergency percutaneous coronary intervention (PCI). A total of 70 patients who accepted emergency PCI treatment were randomly divided into tirofiban and control groups. We determined GMP-140 and troponin I (cTnI) levels before and 12 h after surgery, as well as N-terminal pro-brain natriuretic peptide levels 1 and 7 days after surgery in the two groups. The results showed that GMP-140 and cTnI levels were significantly (P < 0.01) lower in the tirofiban group than in the control group 12 h after operation (17.99 ± 1.01 vs 24.56 ± 1.96 μg/L and 50.96 ± 2.20 vs 58.69 ± 2.34 ng/mL, respectively). The D-value of the N-terminal pro-brain natriuretic peptide levels between 1 and 7 days after operation was significantly higher in the tirofiban group than in the control group (894.19 ± 90.91 vs 829.50 ± 84.18 pg/mL; P < 0.01). The intracoronary application of tirofiban during emergency PCI clearly reduced the GMP-140 level, inhibited the activation function of platelets, improved myocardial perfusion, and helped recover cardiac function in patients.

Comparative Cellular Studies of the VWF Exon 4–5 Deletion Mutation Using Patient-Derived Blood Outgrowth Endothelial Cells (BOEC) and Megakaryocytes

AbstractAbstract 1076The von Willebrand factor (VWF) exon 4–5 deletion mutation has been described in up to 33% of type 3 von Willebrand disease (VWD) patients. The deletion of exons 4 and 5 abolish the first N-linked glycosylation site, which would most likely disrupt trafficking of the protein to the proper chaperones. Since VWF is synthesized in endothelial cells and megakaryocytes, these cells are the most representative of the pathophysiology of VWF mutations. The objective of the current study was to investigate the molecular basis of the exon 4–5 deletion mutation using two comparative cellular systems: blood outgrowth endothelial cells (BOEC) and megakaryocytes (MK). A family in the Canadian type 3 VWD population was identified as having the exon 4–5 deletion mutation. The index case (IC), T001, has type 3 VWD (VWF:Ag=0.01 IU/ml, VWF:RCo=0.02 IU/ml, FVIII:C=0.02 IU/ml, bleeding score=23) and is homozygous for the deletion and the mother, T156, has type 1 VWD (VWF:Ag=0.14 IU/ml, VWF:RCo= 0.13 IU/ml, FVIII:C=0.45 IU/ml, bleeding score=12) and is heterozygous. Peripheral blood samples were obtained from both patients in order to isolate and culture BOEC. Endothelial cell phenotype was confirmed by flow cytometry. VWF expression in patient BOEC showed a decrease in secretion by 87% in T156 and by 98% in T001 compared to control BOEC, similar decreases to those seen in previously published in vitro studies of this mutation. Confocal immunofluorescence (IF) microscopy showed both qualitative and quantitative defects in Weibel-Palade body (WPB) formation in T156 and a complete absence of WPBs in T001, with only diffuse staining of VWF present. Decreased stimulated release of VWF using PMA from T156’s BOEC (26% increase) compared to control BOEC (40% increase) was observed. In T156, this degree of release is similar to the response clinically upon DDAVP administration (1 hour post VWF:Ag=0.30 IU/ml, FVIII:C=1.0 IU/ml; 2 hours post VWF:Ag =0.16 IU/ml, FVIII:C=0.76 IU/ml). Patient MKs were also isolated from peripheral blood samples and cultured by stimulating immunomagnetic isolated CD34+ cells. In T156 the VWF IF staining pattern was both diffuse and punctate and there was some co-localization with P-selectin. Despite the complete absence of VWF in platelets from T001 per immunoblot, VWF was observed in the MKs using IF microscopy. The observations that the patient’s VWF IF staining pattern was diffuse rather than punctate combined with absent colocalization with the alpha granule membrane protein P-selectin within MKs, suggest that MK-synthesized VWF is not packaged into alpha granules. In conclusion we have further evaluated the molecular pathogenesis of a common and recurrent VWD mutation using two comparative cellular approaches, which demonstrates the importance of such cell types in further understanding the pathophysiology of VWD. Disclosures:Walker:Baxter Corporation: Research Funding. James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.

Platelet α-granules: Basic biology and clinical correlates

alpha-Granules are essential to normal platelet activity. These unusual secretory granules derive their cargo from both regulated secretory and endocytotic pathways in megakaryocytes. Rare, inheritable defects of alpha-granule formation in mice and man have enabled identification of proteins that mediate cargo trafficking and alpha-granule formation. In platelets, alpha-granules fuse with the plasma membrane upon activation, releasing their cargo and increasing platelet surface area. The mechanisms that control alpha-granule membrane fusion have begun to be elucidated at the molecular level. SNAREs and SNARE accessory proteins that control alpha-granule secretion have been identified. Proteomic studies demonstrate that hundreds of bioactive proteins are released from alpha-granules. This breadth of proteins implies a versatile functionality. While initially known primarily for their participation in thrombosis and hemostasis, the role of alpha-granules in inflammation, atherosclerosis, antimicrobial host defense, wound healing, angiogenesis, and malignancy has become increasingly appreciated as the function of platelets in the pathophysiology of these processes has been defined. This review will consider the formation, release, and physiologic roles of alpha-granules with special emphasis on work performed over the last decade.

Effects of salvianolic acid B on cardiovascular endothelial cells and platelet activation in a rabbit model of ischemia-reperfusion

To investigate the effects of salvianolic acid B (SA-B) on cardiovascular endothelial cell function and platelet activation during myocardial ischemia-reperfusion in rabbits. A total of 24 New Zealand white rabbits were randomly divided into sham-operated group, ischemia-reperfusion group (untreated group) and SA-B group. The hearts of rabbits in untreated group and SA-B group underwent half an hour of left anterior descending coronary artery (LADCA) occlusion via ligation technology, which was followed by 4 hours of reperfusion to prepared ischemia-reperfusion injury model in vivo. For sham-operated group, the animals were not subjected to occlusion of LADCA. In SA-B treatment group the rabbits were intravenously administered SA-B immediately after LADCA occlusion, and the other two groups were given normal saline in the same way instead of SA-B. The jugular vein bloods of animals were collected before LADCA ligation, half an hour after ligation and after 1-, 4-hour reperfusion, respectively. The content of plasma nitric oxide (NO) was determined by nitrate reductase process. Radioimmunoassay was applied to detect the endothelin (ET) content in plasma and the count of alpha-granule membrane protein-140 (GMP-140) on platelet surface to identify the activation of the platelet. No significant difference was observed before and after sham LADCA occlusion in sham-operated group in the contents of NO and ET in plasma (P>0.05), neither was the count of GMP-140 on platelet surface (P>0.05). The content of NO in plasma detected 0.5 h after LADCA occlusion was significantly decreased in untreated group compared with the sham-operated group at the corresponding time, and they were also much lower than that before LADCA occlusion in the sham-operated group (P<0.05). The plasma content of NO in untreated group showed a progressive decrease in response to the myocardial reperfusion. However, the content of ET in plasma and the count of GMP-140 on platelet surface were remarkably increased after myocardial ischemia as compared with those before LADCA ligation and those detected in sham-operated group (P<0.05). The content of ET and the count of GMP-140 in the untreated group were further increased corresponding to the aggressive reperfusion. The content of NO was significantly increased while the content of ET and the count of GMP-140 were both significantly decreased in SA-B group as compared with untreated group after 1- and 4-hour myocardial reperfusion, respectively (P<0.01). The results show that endothelial dysfunction and platelet activation occur during ischemia-reperfusion in rabbit hearts in vivo and SA-B protects cardiovascular endothelium cells against ischemia-reperfusion injury and inhibits the activation of platelet during myocardial ischemia and reperfusion.

Effects of aerobic exercises therapy on platelet function in phase I essential hypertension

Objective To study the effects of aerobic exercises on platelet function in patients with phase Ⅰ essential hypertension (EH). Methods Low-intensity aerobic exercises therapy was given to 36 patients with phase Ⅰ EH for three months. Changes of their blood pressure, heart rate, platelet aggregation rate (PAGTmax), alpha-granule membrane protein (GMP-140) and mean platelet volume (MPV) werer ecorded and compared to those in 36 normal healthy controls. Results As compared to those in controls,PAGTmax, level of GMP-140 and MPV were all increased in patients with phase Ⅰ EH before exercises therapy(t=15.221, 10. 916 and 16. 401, respectively, all P 0.05). Conclusions Aerobic exercises therapy could be effective in lowering blood pressure in patients of mild EH with increased platelet activation, as well as inhibition ontheir platelet activation. Key words: Aerobic exercise; Essential hypertension; Platelet activation

Level of alpha-granule membrane protein (CD62P) in coronary heart disease

To investigate the role of alpha-granule membrane protein (CD62P), marker of plate activation, in the patients with coronary heart disease (CHD) and to investigate the relationship between the severity of coronary artery stenosis and platelet activation. Peripheral blood samples were collected from 70 patients with CHD, including 28 cases of stable angina pectoris (SAP), 26 cases of unstable angina pectoris (UAP), and 16 cases of acute myocardial infarction (AMI), all of which underwent coronary arteriography, and 20 angiographically normal persons as no-CHD controls. The CD62P level was analyzed by flow cytometry (FCM). Coronary arteriography showed positivity in 70 of the 90 CHD patients. The CD62P positive rate of the UAP group was (28.3 +/- 20.1)%, significantly higher than those of the SAP group [(12.8 +/- 13.3)%] and no-CHD group [(9.9 +/- 6.4)%, both P < 0.05]. The CD62P positive rate of the double vessel disease group was (24.0 +/- 17.8)%, not significantly different from that of the three-vessel disease group [(22.6 +/- 20.4)%], but significantly higher than that of the single-vessel disease group [(11.1 +/- 8.5)%] and that of the no-CHD group [(9.9 +/- 6.4)%, both P < 0.05]. The platelet alpha-granule membrane protein CD62P level is higher in the UAP patients. It is related to the pathophysiological process of UAP and the severity of coronary artery stenosis.

Combination of western and Chinese traditional medicine in treatment of chronic obstructive pulmonary disease with acute exacerbation

Eighty patients with acute exacerbation of chronic obstructive pulmonary disease(COPD)complicated by pulmonary artery hypertention(PAH)were recruited in the study. All patients took conventional western medicine, among them 40 also received injection of Chinese herb medicine Shenxiong(study group)for 14 days. Mean pulmonary artery pressure(mPAP), plasma platelet alphagranule membrane protein(GMP-140), thromboxane B2(TXB2)and 6-keto-prostaglandin F1α(6-K-PGF1α)were measured before and after treatment in both groups. Compared with pretreatment levels and levels in control group mPAP, GMP-140 and TXB2 levels in study group were significantly decreased, while the levels of 6-K-PGF1α were significantly increased. The results indicate that Shenxiong injection may inhibit platelet activation and reduce mPAP. Key words: Lung disease,obstructive; Anticoagulants(TCD)

Influence of O2‐carrying plasma hemoprotein “albumin‐heme” on complement system and platelet activation in vitro and physiological responses to exchange transfusion

Recombinant human serum albumin (HSA) including the synthetic iron(II)-porphyrin (FeP), albumin-heme (HSA-FeP), is a unique O(2)-carrying plasma hemoprotein as a red blood cell substitute. We have investigated the possible influence of HSA-FeP on the complement system and platelet activation in vitro. The amounts of the serum complement titer CH(50) and terminal complement complex SC5b-9 of human blood serum, incubated with HSA-FeP (10, 20, and 40 vol %), were almost the same as those of the corresponding samples with HSA. The effect of HSA-FeP on the platelet reactivity has been demonstrated by conformational changes in the membrane glycoprotein IIb/IIIa and surface expression of an alpha-granule membrane protein P-selectin. Platelet activation in response to the ADP-stimulation was not influenced by the presence of HSA-FeP. It can be concluded that the albumin-heme solution does not facilitate the immunological reaction and platelet activation. Moreover, a 20% exchange transfusion with HSA-FeP into anesthetized rats has been performed to evaluate the circulation and blood parameters for 6 h. Time course changes in all parameters showed features identical to the control group (without infusion) and HSA group.

Platelet Pathology in GATA-1 Dyserythropoietic Macrothrombocytopenia.

Germline mutations in the X-linked hematopoietic transcription factor, GATA-1, have been associated with dyserythropoietic anemia (DEA), macrothrombocytopenia (MTP), and congenital erythropoietic porphyria. Recently, morphological features suggestive of the Gray Platelet Syndrome (GPS) were described in a pedigree with a germline R216Q GATA-1 mutation (Blood 106:6a, 2005). The present study has evaluated platelets (Pl) in the electron microscope from members of a previously described pedigree (Blood 98:2681–2688, 2001) with GATA-1 G208S, MTP, and dyserythropoiesis without anemia, for whom detailed platelet morphology studies have not been reported. The presence of a hemizygous GATA-1 mutation was confirmed by conventional fluorescent dye chemistry sequencing of DNA from patient peripheral blood mononuclear cells. Their Pl revealed wide variations in size, shape, internal and external structure. Some normal sized and giant Pl contained usual numbers of alpha granules and dense bodies, while most of the large cells were hypogranular. Some contained no membrane systems or organelles. Others were filled with masses of dense tubular system (DTS) channels, and others contained the tubular inclusions found in the Medich Giant Platelets Disorder (Platelets 15:345–353, 2004). Many of the hypogranular cells contained small vacuoles that may have been enclosing membranes of alpha granules. Except for the tubular inclusions, all of these structural variations are observed in GPS Pl (Am. J. Pathol. 95:445–462, 1979). However, additional striking abnormalities were seen in GATA-1 Pl that are not characteristic of GPS Pl. Many GATA-1 Pl contained unusual, closely associated dense double membranes differing from normal elements of rough endoplasmic reticulum, smooth endoplasmic reticulum, DTS or surface connected open canalicular system (OCS), but resembling channels of the OCS after exposure to EDTA. They were observed previously only in megakaryocytes (Mk) from one family with GATA-1 DEA-MTP (Nat. Genet , 24:266–270, 2000). The dense double membranes were often found in parallel association and, in some examples, isolating areas of cytoplasm. However, the isolated areas were not undergoing autophagic degradation as such areas enclosed by elements of the DTS do in White Platelet Syndrome Pl (Platelets 15:173,2004). Instead, they proved to be another unique feature of GATA-1 platelets, the presence of Pl within Pl. The sequestered cells were usually mature in appearance and often discoid in form with circumferential coils of microtubules. In some GATA-1 Pl there were two Pl within the same cell, and on rare occasions, a Pl within a Pl within a Pl. Pl within Pl have never been observed previously in any human platelet disorder. Another unique feature, related to Pl within Pl, was the frequent Pl to Pl surface attachment of non-activated cells forming large macrothrombocytes with no similarity to aggregates of stimulated normal platelets.Conclusion: The substructural abnormalities of Pl from patients with germline GATA-1 mutations share some overlap with those seen in GPS Pl, but are distinct and unique. The MTP, Pl within Pl and surface Pl to Pl attachments suggest a major defect in the formation and separation of GATA-1 Pl from pro-Pl of the parent Mk, resulting in the MTP characteristic of the disorder.

Effect of atorvastain on plasma levels of nitric oxide and alpha-granule membrane protein in patients with acute cerebral infarction

目的 探讨阿托伐他汀对急性脑梗死患者血浆NO及血小板α颗粒膜蛋白(GMP-140)水平的影响.方法94例急性脑梗死患者随机分为治疗组(阿托伐他汀组)(48例)和对照组(46例),采用硝酸酶还原法和双抗体夹心酶联免疫吸附法分别测定治疗组和对照组治疗前及治疗4周后血浆NO及GMP-140含量,并同时按照欧洲卒中评分(ESS)标准进行评分.结果治疗后治疗组和对照组血浆NO及GMP-140水平均较治疗前明显下降(P＜0.01),且治疗组较对照组下降明显(P＜0.05);治疗组和对照组ESS分值明显高于治疗前(P＜0.01),但治疗组和对照组的ESS分值无明显差异(P＞0.05).结论阿托伐他汀能降低急性脑梗死患者血浆NO及GMP-140的水平,从而减轻缺血性脑损伤,起到保护脑组织的作用。

Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio‐material for platelet‐rich plasma preparation

Thrombin is commonly used for activating the platelets and releasing the growth factors on the application of platelet-rich plasma (PRP). We have previously reported that chitosan can enhance rabbit platelet aggregation. In this study, the effects of chitosan on the subsequent growth factors release after human platelets activation were examined to evaluate the possibility of chitosan being used as a substitute for thrombin during PRP preparation. Human platelet activation was determined by aggregation, adhesion and alpha-granule membrane glycoprotein expression. Platelet aggregation was measured by the turbidimetric method, the adhesion was directly examined on chitosan-coated glass plates under light microscope and scanning electron microscope (SEM), and the alpha-granule membrane glycoprotein was detected by fluorescent isothiocyanate (FITC)-conjugated anti-CD61 antibody through flow cytometry. The subsequent epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets were assayed by ELISA after mixing with chitosan. The enhancing effects on the platelet adhesion and the aggregation from chitosan were observed. Under both microscopes, the adhesive platelets on the chitosan-coated plates were not only greater in number but also earlier in activation than those on the control plates. With flow cytometry, increased glycoprotein IIIa expression in platelets was detected after chitosan treatment. Greater concentrations of growth factors were measured from PRP after chitosan treatment than after the solvent treatment. Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, we suggest that chitosan may be an appropriate substitute for thrombin in PRP preparation.