The Impact Of Platelets Count On Von Willebrand Factor And ADANTS13Activities In Patients With Immune Thrombocytopenia

Objective: Immune thrombocytopenia (ITP) is a common kind ofautoimmune disease in the blood system. Its incidence is about5-10permillion in Europe and the United States. There are two characteristics for ITP,in which, one is the decrease of platelet count in peripheral blood, the other isthe maturation block of bone marrow megakaryocyte. What is the mainlyperformance is the hemorrhage of mucocutaneous, but visceral bleeding canalso occur in seriously ill patients. In theory, platelet count should becorrelated negatively to the severity of clinical bleeding. The more low theplatelets count is, the more high the risk of bleeding is. However, we canfound in clinical practice that no obvious bleeding events happen in the mostof ITP patients even if their platelets count is low10×109/L. Therefore, theremay be one or some possible compensatory mechanisms in vivo. Under thecircumstances of severe low platelet count, it can decrease the risk of bleedingby regulating some factors of the hemostatic system and the coagulationsystem in ITP patients.Von Willebrand factor(VWF) is an important hemostasis-relatedprotein, which is mainly synthesized and secreted by vascular endothelial cellsand megakaryocytes in bone marrow. When the VWF pro-protein are formed,they are transported to the endoplasmic reticulum, and at the C end they formdimmers with disulfide bond. Then they are transferred to Golgi apparatuswhere they realize multimerization. At last, the VWF pro-protein becomeVWF multimers which have a large molecular. The mature VWF have veryimportant effects on primary hemostasis and the second phase of hemostasis.On the one hand, when the vascular is injuried and subendothelial collagen isexposed, VWF can mediate the binding of platelets to collagen and thenpromote platelets adhesion and aggregation and result in platelets thrombosis at last. On the other hand, VWF serves as a carrier of FVIII by binding FVIIIwith non-covalen. Therefore, VWF can protect the FVIII not be hydrolyzedand result in the prolong half-life period. Studies have shown that themolecular weight of VWF in normal human plasma is between500-20,000kDa and that its molecular weight is regulated by ametalloproteinases which is called ADAMTS13. The enzyme ADAMTS13plays this role by cleaving the peptide bond of Tyr1605-Met1606in VWF-A2domain. They can maintain VWF existing in normal molecular weight, so that,in our body，there is not thrombosis because of excessive molecular weightand bleeding because of the undersize of the molecular weight. The purpose ofthis study is trying to investigate whether severe decreased platelet countcould decrease the activity of ADAMTS13resulting to the reducing thehydrolysis of VWF by ADAMTS13and increased VWF haemostatic activity,which can prevent bleeding risk resulting from the thrombocytopenia.Methods:1The objects were divided into experiment groups and normal controlgroups.⑴Experiment group: We collected some patients of primary ITP inthe second hospital of Hebei Medical University from Janury2011to June2013. All the patients are consistent with the diagnosis standard of immunethrombocytopenia according to third edition of the diagnosis and treatmentstandard of Hemopathy by editor in chief Zhang Zhinan. No one coexists withthe the hypertension, diabetes, acute and chronic infectious diseases andimmune rheumatic disease. The two groups were further divided topre-treatment group and the after-treatment group.①pre-treatment group: Wetake50patients with primary ITP whose platelet count was less than10×109/Land normal white blood cell and hemoglobin levels beforetreatment.②After-treatment group: We take30patients with ITP which wereacceptted the regular treatment and the platelet count was returning to normal(PLT＞100×109/L) as the after-treatment group.⑵Normal control group: weselected50healthy persons with the normal routine blood test and thecoagulation in the routine examination as normal control group. These persons didn’t have hypertension, diabetes, coronary heart disease, immune rheumaticdisease and cerebrovascular disease. At the same time, they did not take anymedication. It included25men and25women. Their ages were20to69yearsold. The median age was48years old.2Specimen collection: The peripheral venous blood was obtained fromthe patients and normal population. The sodium citrate was used asanticoagulant. The plasma was collected by centrifuging and freezed in-70℃foruse.3Test items: The tests indexes included VWF: Ag、VWF:CB、VWF:CB/VWF: Ag ratio and the activity of ADAMTS13in vitro. VWF: Ag andVWF:CB were detected by ELISA. And FRET-VWF86was used to determinethe activity of ADAMTS13in vitro.4Statistical analysis: Mann-Whitney U was used to examine thedistribution of the measurement data. The data of normal distribution wereexpressed by mean±standard deviation (x±s). One-Way ANOVA was usedto examine multiple groups data which compared with each other, in which,SNK-q test was used in homoscedasticity data and Dunnett’s C test was usedin the data of heterogeneity of variance. P＜0.05was regarded as a significantstatistical difference.Results:1VWF antigen: The ELISA results showed that the plasma levels ofVWF: Ag of the pre-treatment group, after-treatment group and the normalcontrol group were (0.97±0.46),(0.99±0.55) and (0.96±0.41), respectively (P＞0.05). Therefore, there were no obvious differences in VWF: Ag among thepatients with ITP before and after treatment and the healthy people.2VWF:CB: The VWF:CB in the pre-treatment group, after-treatmentgroup and the normal control group were (2.08±0.46),(1.09±0.35) and(1.13±0.41). It was said that the VWF:CB in pre-treatment group wassignificantly higher than that in normal control group and after-treatmentgroup (P＜0.05). But there were no differences between normal control groupand the after-treatment group in VWF:CB (P＞0.05). We concluded that the VWF:CB of patients with ITP (PLT≤10×109/L) in plasma was significantlyhigher than that in persons with normal platelet. But when their platelets countretured to normal, the VWF:CB also retured to the level of normal population.3VWF:CB/VWF: Ag ratio: The ratio of the pre-treatment group,after-treatment group and the normal control group were (2.03±0.67),(0.98±0.58) and (1.10±0.41). It showed that the ratio of VWF:CB/VWF: Ag inpre-treatment group was significantly higher than that in normal control groupand after-treatment group (P＜0.05). But the ratio of VWF:CB/VWF: Ag inafter-treatment group had no differences with normal control group (P＞0.05).It is concluded that the ratio of VWF:CB/VWF: Ag in patients with ITP(PLT≤10×109/L) was significantly higher than that in persons with normalplatelets count. But when their platelets count returned to normal, theVWF:CB/VWF: Ag ratio returned to the level of normal population.4ADAMTS13activity in vitro: The ADAMTS13activity in vitro ofpre-treatment group, after-treatment group and normal control group was(612.6±85.58),(632±84.28) and (651±82.59) detected by FRET-VWF86(P=0.312, P＞0.05).There were no significant differences among the threegroups. Therefore, the activity of ADAMTS13in vitro of ITP patients inpre-treatment group with severe decreased platelets count and after-treatmentgroup with normal platelets count after treatment both had no differenceswhen compared with normal population.Conclusions:1There had no obvious changes in VWF: Ag and activity ofADAMTS13in vitro in ITP patients with decreased platelet and healtypopulation, but the VWF:CB and VWF:CB/VWF: Ag ratio were significantlyhigher than that in normal population. It is indicated that the cleavage activityof VWF by ADAMTS13was reduced by decreasing platelet count inpatients with ITP in vivo. There were no changes in activity of ADAMTS13itself.2For the immune thrombocytopenia patients, molecular weight of VWFmultimer was raised through reducing the activity of cleavage of ADAMTS13 on VWF and it could raise the hemostatic activity furtherly. So it could be oneof the compensatory mechanisms to reduce the risk of bleeding in patientswith ITP.3Through the model of ITP patients, it was firstly confirmed that thecleavage activity of ADAMTS13on VWF was adjusted by the changes ofplatelet count in vivo. So the discovery that cleavage activity of ADAMTS13on VWF could be promoted by platelets was enriched and perfected furtherlyby this study.