| BackgroundHemophilia A is an X chromosome linked recessive hereditary hemorrhagic di sease.Statistics show its incidence for 1/10000, the sever type 1/16000,about 350 thousand hemophlia patients globle there is according to the study of W HO and WFH.Estimated 10-13 million cases in China.The pathological basis of hemophilia A is a genetic defect in coagulation factor VIII (FVIII) leads to the factor VIII level (FVIII:C) and (or) function to reduce the blood does not clot properly. The main clinical manifestations of spontaneous joint and t issue bleeding and bleeding caused by the deformity.Currently, we defined the clinical type according to plasma FVIII/F IX activity, for sub clinical typ e, mild, moderate and sever type.Statistics show that up to 60-70% are sev er type for the mild and moderate cases misdiagnosis rate is high.In untre ated cases, severe hemophiliacs bleed 1-6 times a month on average,spontane ous joint, muscle and soft tissue bleeding occur, recurrent joint bleeding caused by joint synovitis, cartilage destruction, osteoarthritis, muscle atrophy pathological changes such as the loss of joint function and disability and chronic haemophilic joint pain and movement disorder reduces the quality of life of patients, in addition to haemophilic arthropathy, osteofascial compartment integrated syndrome, pseudotumor, important viscera hemorrhage for hemophilia patients with common risk of complications.All that decreased in the patient’s quality of life.The bleeding frequency and severity are associated with coagulation factor levels, treatment for bleeding is still with coagulation factor replacement therapy, prophylactic treatment and on-demand treatment become the main strategy to block joint disability. The treatment of recurrent bleeding events and complications has brought a great financial burden to patients.. Medical condition, economic endurance, and drug source are the main factors that restrict the treatment.In clinical work, we found that even if the same plasma clotting factor levels, the severity of the bleeding and bleeding frequency may also exist significant difference. Many studies by monitoring the frequency of bleeding events or infusion of clotting factors and found that about 10-15% severe hemophilia A is characterized by mild hemorrhage (bleeding 6 times/year or less), the phenomenon is called the clinical heterogeneity of severe hemophilia, Also found that reported in the foreign literature in Van Dijk, K, if without treatment, some patients in bleeding events up to 96 times, and also some with very little bleeding, this phenomenon called severe hemophilia the clinical heterogeneity Our previous work has also confirmed that the heterogeneous phenomenon exists, Li analysised 223 cases of severe patients’ data, it is found that in South China the severe hemophilia A patients clinical manifestation has obvious heterogeneity, about 12.9% with mild phenotype; about 9.3% above 15 without joint deformity.The mechanism of this clinical heterogeneity is not clear so far, although hemophilia is a single gene mutations genetic disease, but its clinical manifestations is result of genetic and environmental factors, single gene phenotype cannot explain the severe patients with mild bleeding phenotype phenomenon.In Santagostino’s research, the author detected the thrombin generation test for the patients with severe hemophilia, to promote coagulation and anticoagulation activity, confirmed that patients with severe clinical manifestations of heterogeneous and procoagulant and anticoagulant activity differences related, prompts FVIII activity is not the only factor to measure clinical type, also shows that the plasma levels of coagulation factors and can not be completely accurate measurement and prediction in patients with bleeding risk and treatment needs and long-term prognosis. Other may still exist on clinical manifestations produced factors worthy of our inquiry.Combined with the existing domestic and foreign research results and our previous work we found that effects of severe hemophilia A’s clinical heterogeneity may be (1) FVIII gene mutation type; (2) promoting coagulation and anticoagulation protein activity related factors (such as F VIII drug pharmacokinetics and vwfag level, plasminogen activity); (3) hemorrhage symptoms related factors (such as the initial age of bleeding, the first joint bleeding age, joint function); (4) other factors such as physical activity level, body mass index, vasoactive and psychological state related .platelets play a vital role in coagulation,. Cambien Beatrice et consider that P-slectin can promote hemostasis by promoting platelet aggregation and fibrin formation. Van Bladel et al found that the activity of platelets in patients with mild and moderate hemophilia A was significantly higher than that of patients with severe severe. After platelet activation, the plasma membrane was formed to form platelet microparticles, the surface of the membrane was exposed, and a large number of coagulation factors was provided to promote thrombin generation and coagulation. As the product of activation, the platelet particles expand the activation reaction and transfer the information to the distant, and increase the activation level of the distant platelets.Hrachovinova I et al confirmed that P-interacts with its ligand PSGL-1 and promotes the generation of in mice.. We assume that clinical heterogeneity of patients related to platelet activation, and the level of PMPs. The clinical manifestations impacts the coagulation process.Our previous work screening analysised 223 patients with severe hemophilia A of our department and found indeed exists heterogeneity. This study intends to study the platelet activation function and platelet derived microparticles (PMPs) of two groups of severe hemophilia A (severe hemophiliaA with mild phenotype and severe hemophilia A with severe phenotype).and PMPs under different conditions and to find mechanism of the relationship between MPS and clinical heterogeneity.MethodsPart I. platelet count, platelet morphology, platelet activation function.1.1 Case Information:The severe HA was defined of FⅧ:C<2% according t o Bethil TC program. Case Information:The severe HA was defined of FⅧ: C≤2% according to Bethil TC program,we selected register from NanFang h osipital affiliated to southern medical university with a complete medical histo ry data of a total of 367 patients with severe hemophilia A, eliminate FⅧ in hibitor positive patients, after clinical informed,31 patients met the inclusion, severe phenotype 21 cases, mild group 10cases, healthy adult male volunteer s 17.Bleeding frequency 6 times/year,24 times/year as the cut-off point, defined patients with spontaneous in bleeding frequency is less than or equal to 6 times/year in the past 2 years for mild severe hemophilia and> 24 times/years for severe phenotype , The age and weight of each group were matched. Subject BMI in normal range.1.2 main instruments and reagents:1.2.1 main instruments:thermo high-speed centrifuge (thermo, USA) and flow cytometry detector (CantoII, American Becton Diekson), blood cells XE-2100M analysis water system (Sysmex Shanghai).1.2.2 main reagent:mouse IgG1-PE (American Becton Diekson company), CD41a-APC (American Becton Diekson company), CD42b-PE (American Becton Diekson company), CD61-FITC (American Becton Diekson company), CD62P-PE (American Becton Diekson company), CD63-PE (American Becton Diekson company),1% poly formaldehyde, PBS phosphate buffer (the Institute from the distribution).1.2.3 other:Falocn sample tube (American Becton Diekson company) and micro pipettes (Eppendorf, Germany), tube vacuum hemostix, citric in 0.109mol/L acid sodium anticoagulation, ethylenediaminetetraacetic acid (EDTA) anticoagulation tube, disposable blood sampling needle.1.3 experimental procedure and method:Cytometry flow (FCM) was used to detect the positive rate of platelet membrane surface CD62P and CD63 in platelet rich plasma (PRP).1.3.1 specimen processing:Patients for over 72 hours of washout period (test before 72 hours in no bleeding events, useless medicine records) after blood sampling, in accordance with the venous blood collection guidelines, on an empty stomach in the morning, with more than 20 gauge needle disposable blood taking needle acquisition elbow anterior vein venous blood, in order to avoid causing additional platelet activation process of blood in the prohibition of the use of a tourniquet, blood sampling needle is inserted in EDTA anticoagulant tubes, before 2 ml venous blood was used for detection of platelet count, continuing to collect 2.7 ml venous blood along the wall of the test tube slowly into the 0.109mol/L sodium citrate as anticoagulant tube (blue cap tube), avoid foaming blood, blood and anticoagulant ratio of 9:1.30 minutes to deal with blood samples, the first blood samples 800rpm centrifuge for 5 minutes, draw on the upper plasma, get rich blood platelet plasma (PRP) after the sample treatment with the following sample.1.3.2 monoclonal fluorescent antibody labelingEach sample were taken 4 sample tubes, as above were added to the fluorescently labeled with McAb was IgG1, CD61 and CD62P, CD63 monoclonal antibody and gently mix, at room temperature to avoid light incubation for 20 minutes, fully . integrated with the antibody and cell surface marker,20 minutes later add 5 ml of PBS pH 7.43000 RPM/separation of the heart 5 minutes, remove excess unbound monoclonal fluorescent antibody, with 1% paraformaldehyde 0.5ml diluted immediately machine testing.Part II.PMPs detection by FCM,.2.1 Case Information:The severe HA was defined of FⅧ:C≤2% according t o Bethil TC program. Case Information:The severe HA was defined of FⅧ: C≤2% according to Bethil TC program,we selected register from NanFang h osipital affiliated to southern medical university with a complete medical histo ry data of a total of 367 patients with severe hemophilia A, eliminate FⅧ in hibitor positive patients, after clinical informed,31 patients met the inclusion, severe phenotype 21 cases, mild group 10cases, healthy adult male volunteer s 17.Bleeding frequency 6 times/year,24 times/year as the cut-off point, defined patients with spontaneous in bleeding frequency is less than or equal to 6 times/year in the past 2 years for mild severe hemophilia and> 24 times/years for severe phenotype,The age and weight of each group were matched. Subject BMI in normal range.All the samples are for PMPs det ection.11 sever type patients agreed we had venous blood collected for PMPs detecti on.Tthe baseline state, acute hemorrhage, after factor VIII injection treatmen t. Baseline samples were collected within 72 hours without bleeding, useless medicine records meeting the; acute bleeding blood restrictions in bleeding 1 hour after the incident in and before blood sampling without the use of coag ulation factor; factor VIII injection (dose 15 IU/kg) phlebotomy in 8-12 hours after the direct factor. By comparing by subjects at baseline, acute hemorrhag ic period, factor in PMPs level understanding of the severe phenotype in diffe rent stages of patients with peripheral blood PMPs level and factor substitutio n treatment effect on the level of PMPs.2.2 main instruments and reagents:2.2.1 main instruments:thermo high-speed centrifuge (thermo, USA) and flow cytometry detector (CantoII, American Becton Diekson), blood cells XE-2100M analysis water system (Sysmex Shanghai).2.2.2 main reagentmouse IgGl-PE (America, Becton Diekson company), CD41a-APC (America, Becton Diekson company), 1μm standard fluorescent beads (Sigma-Aldrich products, University of Hong Kong presented),1% poly formaldehyde, PBS phosphate buffer (the Institute from the distribution).2.2.3 other:Falocn sample tube (American Becton Diekson company) and micro pipettes (Eppendorf, Germany), tube vacuum hemostix, citric in 0.109mol/L acid sodium anticoagulation, ethylenediaminetetraacetic acid (EDTA) anticoagulation tube, disposable blood sampling needle.2.3 experimental procedure and method:2.3.1 method:Patients after more than 72 hours of washout period (test before 72 hours in no bleeding events, useless medicine records) after blood sampling, in accordance with the guidelines for venous acquisition, fasting, with more than 20 gauge needle disposable blood taking needle acquisition elbow anterior vein venous blood, in order to avoid causing extra platelet activation process of blood in the prohibition of the use of tourniquet. The blood sampling needle is inserted into the EDTA anticoagulant tubes, before 2 ml venous blood was used for detection of platelet count, continuing to collect 2.7 ml venous blood along the wall of the test tube slowly into the 0.109mol/L sodium citrate as anticoagulant tube (blue cap tube), avoid foaming blood, blood and anticoagulant ratio of 9:1. After 30 minutes with blood, the blood 800rpm centrifugation for 5 minutes, learn from the upper plasma, get rich platelet rich plasma (PRP), the rich plasma platelet high-speed centrifugal, 20 minutes 1550g 20 DEG C centrifugal, full removal of platelets, left the supernatant,18000g 20 DEG C centrifugal 30 minutes supernatant. Frozen at-80, and the subsequent unified flow cytometry for particulate detection.The experimental process of the collected specimens of 37 DEG C for rapid rewarming and platelet specific fluorescent antibody CD41-FITC marker PMPs, using standard microspheres for positioning control and detection conditions of the machine settings, adjusting machine threshold, gate count and PMPs accounted for percentage of 1 mu mCD41 positive granules detected static bearing state under severe hemophilia patients with different clinical phenotype and normal control group, the level of PMPs, continuous low dose f VIII prevention and treatment of severe phenotype of the patients at baseline and acute hemorrhage treatment period and after 3 months of PMPs level.After the addition of sample with room temperature for 30 minutes, the specific binding of the mAb and platelet particles was detected, and the volume of PBS solution was adjusted to 0.5mL. Machine test:fill up and check the sheath liquid barrel, abandoned waste, open the FCM and pressurized instrument gas path, emptying bubbles and calibration of light path of the instrument in the best condition, the variation coefficient< 2%, before the establishment of the angle light scattering on the lateral angle light scattering dual logarithm scattered point graph (FSC LOG-SSC log) and in diameter is 1 m standard microspheres as a positioning control, according to the positioning control, instrument and SSC threshold adjusted to 200, FSC and voltage gain was adjusted to 100 and 1000. Under these conditions, the gathering and analysis to the signal were by PMPs and standard microspheres, and under the same conditions determination of a tube containing only deionized water blank control in order to validate the instrument noise signal and the degree of interference. The PMPs PMP was detected by flow cytometry and the detection zone was set with 1 m. The expression of CD41-PE was detected by flow cytometry.. The particles of the inner (micro diameter of 0-1 m) were analyzed (Fig.2-1). The PMPs was defined as the diameter range of 0-1 m and the surface labeled CD41 positive.Result2.1 general case data comparison:severe hemophilia and severe phenotype group (n = 21) with an average age of (23.55.33+) years, mild phenotype group (n= 10) average annual age (28.97.06) and normal control group (n=17) (31.08\8.11) years, between each age difference was not significant. The severe phenotype Group F VIII activity (FVIII:C) (1.04+0.24)%, two groups of F VIII activity comparison, P values=0.304 (0.96+0.35)%, mild phenotype Group F VIII activity, the difference was not significant.2.2 each platelet count:severe phenotypic group (n= 21) mean platelet count (292.5 +83.69) G/L, mild phenotype group (n= 10) and platelet count (256.3+60.52) G/ L, the normal control group (n= 17), platelet count (239+50.82) G/L, mild phenotype group and normal control groups, P value of 0.3519. No significant difference, severe phenotype and the control group comparison p to 0.369, severe hemophilia different phenotype between groups P=0.6217, difference not significant. See table 1-2.2.3 each platelet CD62P expression rate of severe phenotype group (n= 21) CD62P expression was an average (36.2+9.27)%; mild phenotype group (n= 10) CD62P expression was an average of:(49+11.81)%, and the normal control group (n=17) CD62P expression was an average (22.06+10.72)%. Normal group and mild phenotypic mean comparison, P= 0, the difference was very significant; normal group and the severe phenotype as compared with the control group, P= 0.001, the difference is very significant. Two groups of clinical phenotype of patients with severe hemophilia between group comparisons, P values=0.0408 and difference between significantly.2.4 the expression of CD63 in platelets was compared:severe phenotype group CD63 expression (34.82+7.13)%; mild phenotype group (51.15+14.3)%; normal group CD63 expression was (19.08+17.05)%. Normal group and mild phenotype group, P value= 0, extremely significant difference; normal group and the severe phenotype group, P= 0.04, the difference was significant; clinical phenotype of the two groups of patients with severe hemophilia between groups P 0.0044, the difference was extremely significant.A comparison of baseline PMPs levels in 2.5 groups:Percentage of mild phenotype group PMPs are numbers plus standard deviation is (2.06+1.33)%, severe phenotype group percentage were number plus standard difference (0.95+0.64)%; normal group PMPs percentage mean (1.061.20)%, among the three groups (P value of 0.02, statistical differences between groups significantly.Comparison of PMPs levels in 2.6 patients with severe phenotypes:There are 11 severe phenotype of patients participated in the baseline state, acute hemorrhage and FVIII replacement therapy after PMPs level detection, the income data of paired samples t test. For the baseline level of patients with PMPs (0.85+ 0.18)%; acute bleeding stage PMPs level (6.50+0.51)%; F VIII replacement therapy after levels of PMP s for (0.45+0.40)%, acute hemorrhagic period, baseline, after administration of PMPs base level pairwise paired samples t test p value is 0, difference is very significant.ConclusionAdults with severe haemophilia A mild phenotype in patients with platelet activation marker CD62, CD63 expression is higher than the severe phenotype, and both of them are higher than that of the normal control group, significant difference, prompt severe hemophilia function of platelet activation in patients with the existence of compensatory activation, the activation level differences may be one of the reasons why the clinical phenotype heterogeneity.In severe hemophilia a different clinical phenotypes in resting state in patients with PMPs were higher than the normal population and mild phenotype of PMPs count higher than the severe phenotype, conclusion and different clinical phenotypes of severe hemophilia A patients with platelet activation in a consistent state that PMPs also involved in patients with hemophilia a compensatory procoagulant, is one of the reasons for the patients with severe clinical manifestations of heterogeneity and platelet microparticles may can be used as a predictor of prognosis of patients with severe clinical phenotype. Acute hemorrhage and F after injection of PMPs changes more importance of procoagulant hemostatic function in PMPs. |
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