Study On The Haemostatic Mechanism Of Hemocoagulase Atrix And On Two Patients With Phytosterolemia

Part ⅠIn Vitro Effects of Hemocoagulase Atrix and its Compounds onBlood CoagulationSnake venoms are complex mixtures of biologically active proteins and peptides. Many ofthem affect hemostasis by activating or inhibiting coagulant factors or platelets, or bydisrupting endothelium. A lot of Sake venoms has been applied to diagnose and treathemorrhagic or thrombotic disease. Like thrombin, TLEs specifically catalyse limitedcleavage of fibrinogen and lead to clotting of fibrinogen. Based on the rates of release offibrinopeptides A and B, TLEs can be classified into three groups, venombin A, venombinB and venombin AB. Hemocoagulase Atrix is isolated from Bothrops atrox venom, mainlycontained of bathroxobin and little of factor Ⅹ activator. Bathroxobin do not usuallyactivate FXIII, but cleave only fibrinopeptide Aαchain at one position(Arg16-Gly17).This results in formation of abnormal,“loose” fibrin clots composed of short polymersnot stabilized by transglutaminase-catalyzed crosslinking. F Ⅹ activator promotesthrombin generation and FⅩⅢ activation through activating FⅩ, and then form compactfibrin. Hemocoagulase Atrix has been widly used in treatment of bleeding neurosurgery,gynecology, orthopaedics，and in treatment of alimentary or respiratory tract hemorrhage.As yet there is no In vitro study on the thereapy of hemorrhagic disease.Objective: To investigate the procoagulation effects of Hemocoagulase Atrix and itsCompounds (batroxobin and factor Ⅹ activator) on plasma of normal subjects and patientswith bleeding disorders and their mechanisms.Methods: Activated partial thromboplastin time (APTT) and prothrombin time (PT)were measured. The factor (F) Ⅹ activation and thrombin generation were analyzed byusing chromogenic substrate method. Results: The plasma APTT of normal subjects was shortened in dose-dependentmanner by Hemocoagulase Atrix, batroxobin and FⅩ activator, and the prolonged APTTof plasma from patients with bleeding disorders could be corrected, but PT showed nogreat changes. FⅩ activator could promote FⅩ activation and thrombin generation, whileneither Hemocoagulase Atrix nor batroxobin showed such abilities.Conclusion: These results of our study indicate that Hemocoagulase Atrix promotecoagulation process, and correct coagulation abnormalities in patients with bleedingdisorders. Its main compound batroxobin directly acts on fibrinogen, and FⅩ activatorpromotes thrombin generation through activating FⅩ. Part Ⅱ Study on the Clinical Features and Gene Diagnosis of phytostero-lemia Causing to Macrothrombocytopenia and hemolytic anemiaPhytosterolemia (sitosterolemia) is a rare autosomal recessive lipid storage disease.Up to now, there are only about100cases reported worldwide. Patients suffering fromphytosterolemia present primarily with tendon and tuberous xanthomas and prematurecoronary heart disease, sometimes accompanied by arthralgia and arthritis. Apart from theintrinsic reasons of platelet, lipid dysbolism can be associated with thrombocytopenia.Recently, we found2patients from2unrelated families who presented withmacrothrombocytopenia and hemolytic anemia, increased plasma levels of phytosterols,and had homozygous mutations in ABCG5or ABCG8genes. Investigation of clinicalcharacteristics identification of gene mutations would be essential for correct diagnosis andproper treatment of phytosterolemia with macrothrombocytopenia and stomatocytosis.Objective: To study the clinical features and ABCG5/ABCG8gene mutations of twopatients of phytosterolemia involved in macrothrombocytopenia and hemolytic anemia.Methods: Erythrocyte and platelet morphology was examined under light microscope;Platelet glycoproteins (GP)(Ib/IX and IIb/IIIa) were analyzed using flow cytometry;Platelet aggregation tests were performed with aggregometer; Plasma phytosterols weremeasured by high pressure/performance liquid chromatography (HPLC) method; All of ABCG5and ABCG8exons and intron-exon boundaries were amplified by PCR anddirectly sequenced to identify mutations. In order to exclude the probability ofpolymorphism, the corresponding gene mutation sites of four family members and healthyindividuals were also analyzed by PCR-RFLP.Results: The two patients presented with macrothrombocytopenia and hemolyticanemia, xanthomatosis. Plasma concentrations of phytosterols, especially sitosterol, weremarkedly elevated (30fold) in the affected patients. Two novel mutations，ins12283C andISV7+3G>A were identified.Conclusions1. Two phytosterolemia patients were identified, and all of them associated withmacrothrombocytopenia and hemolytic anemia; Two novel mutations were found firstly:ins12283C and ISV7+3G>A.2. The patients with phytosterolemia might exclusively present with hematologicabnormalities, patients with macrothrombocytopenia and/or hemolytic anemia should beincluded in differentiation from phytosterolemia.3. When the patients with thrombocytopenia had no response to conventionalmanagements, the shapes of red cells and platelets should be examined and phytostero-lemia should be considered if prominent macrothrombocytopenia and stomatocytosiscoexist.