Comparison of the changes in platelet membrane glycoproteins between patients with hemorrhagic thrombopathy and healthy people

Lin SHEN; Furong LU; Di SHEN

ABSTRACT

BACKGROUND:

It has been confirmed that platelet aggregation defect presents in patients with hemorrhagic thrombopathy, and platelet membrane glycoproteins play a significant role in the process of platelet aggregation.

OBJECTIVE:

To observe the expression changes of platelet membrane glycoproteins between patients with hemorrhagic thrombopathy and healthy people.

DESIGN:

Case-control analysis.

SETTING:

Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College,Huazhong University of Science and Technology.

PARTICIPANTS:

① Seventy-nine patients with hemorrhagic thrombopathy who received treatment from January 2001 to March 2003 in the Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were enrolled in this study. The patients, including 31 male and48 femlae, averaged (35.76±14.14)years old. They all agreed to participate in this study and met with the diagnosis of hemorrhagic thrombopathy revised in 1996.Informed consents were obtained from all the patients and their relatives.The course of multiple bleeding symptoms was 1 to 14 years. And the bleeding sites were totally 167 extremity petechia was found in 64 cases, rhinorrhagia in 33 cases, hypermenorrhea (more than 150 mL in each cycle) in 29 cases, gingival bleeding in 28 cases, ocular fundus bleeding in 8 cases and conjunctival hemorrhage in 5 cases.②lnclusive criteria All the cases presented clinical manifestation of multiple focal bleeding;The laboratory tests including platelet counts,bleeding time and coagulation profiles were examined with no marked abnormal changes;According to hemorrhage sites, the patients were examined by specialists of internal medicine,otorhinolaryngology, gynecology, stomatology and ophthalmology, and no specific diagnostic focuses of infection were found; They had normal blood pressure and heart rate,and those with thrombocytasthenia had been ruled out through ristocetin test. ③Another 34 healthy volunteer donors, 15 males and 19 females with an average age (30.12±7.14) years,were selected as normal control group.

METHODS:

① Detection of platelet aggregation ratio Adenosine diphosphate (ADP, final concentration 1.90 μmol/L),arachidonic acid(AA, final concentration 0.37 μmol/L),and platelet activating factor(PAF, final concentration 150 nmol/L)were used as aggregation inductors, and Chronolog 430 type aggregometry was used to detect platelet maximum agglutination ratio.When maximum agglutination ratio was within 21％ and 40％, it was considered as aggregation defect, and when below 20％, it was considered as aggregation absence.② Test of platelet membrane glycoprotein 3.6 mL cubital venous blood was drawn from the patients and mixed with 20 g/L disodium ethylenediamine tetraacetic acid(EDTA-NA2) for the purpose of anticoagulation at 19, and the same procedure was performed on the blood samples from the normal control group. The plasma was isolated and collected, after being centrifuged, the supernatant liquid was discarded, and paraform was added to fix the rest of the platelet solution. The platelet concentration was adjusted to 3×107 L-1. 100 μL regulated platelet solution was extracted, and fluorescein isothiocyanate (FITC)-marked CD42b (anti-GP Ⅰ b),CD41 (anti-GP Ⅱ b), CD61 (anti-GP Ⅲ a), CD42b/CD42a (anti-GP Ⅰ b/Ⅸ ), CD41/ CD61 (anti-GP Ⅱ b/Ⅲ a) and CD62p(anti-P-selectin)clonal antibodies 200 μL were added respectively and well mixed,and then cultured for 30 minutes at room temperature away from light. Finally phosphate buffer solution was added to the solution till the volume reached 1 mL,and FACS420 flow cytometer was used to analyze the samples, one sample for 10000 platelets, and the results were demonstrated by positive fluorescence percentage.MAIN OUTCOME

MEASURES:

① Platelet aggregation ratio. ② Expression of platelet membrane glycoproteins.

RESULTS:

All the patients and the healthy subjects were involved in the analysis of results. ①All the cases were found to have platelet aggregation defect or absence which could be induced by single or more platelet aggregating agents. No abnormal platelet aggregations were found in the control group. ② The fluorescence intensities of GPIb/Ⅸ, GP Ⅱ b/Ⅲa complexes, GPIb, GPⅢa and P-selectin in patients with hemorrhagic thrombopathy were lower than those in the healthy subjects (t =2.50-5.57,all P ＜ 0.05). The fluorescence intensity of GP Ⅱb in patients with hemorrhagic thrombopathy was slightly higher than that in the healthy subjects, but no significant difference was found. (t =0.86, P ＞0.05).

CONCLUSION:

Abnormal expressions of platelet membrane glycoproteins may be partial pathogenesis of hemorrhagic thrombopathy.