Structure and function of snake venom proteins affecting platelet plug formation

Many snake venom proteins have been isolated that affect platelet plug formation by interacting either with platelet integrins, membrane glycoprotein Ib (GPIb), or plasma von Willebrand factor (VWF). Among them, disintegrins purified from various snake venoms are strong inhibitors of platelet aggregation. Botrocetin and bitiscetin derived from Bothrops jararaca and Bitis arietans venom, respectively, induce VWF-dependent platelet agglutination in vitro. Several GPIb-binding proteins have also been isolated from snake venoms. In this review, we focus on the structure and function of those snake venom proteins that influence platelet plug formation. These proteins are potentially useful as reagents for the sub-diagnosis of platelet disorder or von Willebrand disease, as well as for clinical and basic research of thrombosis and hemostasis.     

The safety of plasma-derived von Willebrand/factor VIII concentrates in the management of inherited von Willebrand disease

Until the mid-80s, cryoprecipitate has been the mainstay of treatment of patients with von Willebrand disease who were unresponsive to desmopressin. The advent of virally-inactivated factor VIII (FVIII) concentrates containing von Willebrand factor (VWF), originally devoted to hemophiliacs, provided a better therapeutic approach to von Willebrand disease. These VWF/FVIII concentrates were introduced in clinical practice after the positive results obtained in several prospective and retrospective clinical studies. They are safe and can be suitable also for home treatment. Allergic or anaphylactic reactions are limited to the rare patients with deletions of VWF gene. In repeated infusions during surgery, the dosage and timing of administration should be planned to keep FVIII below 150 – 200 U/dl to avoid any possible risk of thrombosis.     

Anti-thrombotic effects and bleeding risk of AJvW-2, a monoclonal antibody against human von Willebrand factor

1. A murine anti-human vWF monoclonal antibody, AJvW-2, was developed that inhibited the interaction between platelet glycoprotein Ib (GPIb) and von Willebrand factor (vWF) during the ristocetin- (IC₅₀=0.7±0.1 μg ml⁻¹) and botrocetin- (IC₅₀=1.8±0.3 μg ml⁻¹) induced aggregation of human platelets.
2. AJvW-2 inhibited the high shear stress (10.8 N m⁻²) induced aggregation of human platelets dose-dependently with an IC₅₀=2.4±0.3 μg ml⁻¹, but had no effect on low shear stress induced platelet aggregation (1.2 N m⁻²) up to 100 μg ml⁻¹.
3. AJvW-2 also inhibited the high shear stress (5.0 N m⁻²) induced adhesion of human platelets to collagen I with the same efficacy (IC₅₀=2.4±0.3 μg ml⁻¹), but had no effect at low shear conditions (1.5 N m⁻²).
4. AJvW-2 inhibited the botrocetin-induced aggregation of platelets from guinea-pig, rat, rabbit, dog and pig at the same concentration range as human platelets; it likewise also inhibited the high shear stress induced aggregation and adhesion to collagen I of guinea-pig platelets.
5. AJvW-2 prevented arterial thrombus formation in guinea-pigs at a dose of 100 μg kg⁻¹ without prolonging the template bleeding time, whereas the GPIIb/IIIa antagonist lamifiban mediated inhibition of thrombosis at 1000 μg kg⁻¹ was accompanied by a significant prolongation of the bleeding time.
6. These results suggest that AJvW-2 is a potent inhibitor of the GPIb-vWF interaction and a potential novel antithrombotic agent with lower bleeding risk than GPIIb/IIIa antagonists.

     

慢性肾功能不全患者血浆von Willebrand因子水平变化及其临床意义

目的研究慢性肾功能不全患者(包括尿毒症血液透析)的血浆yon Willebrand因子(vWF)含量的变化及其临床意义.方法选择43例慢性肾功能不全患者,根据有否血液透析分为透析组22例;非透析组21例进行观察,对照组为20例健康体检者.分别检测各组血浆vWF含量,并对透析组患者血浆vWF含量与透析月数之间;非透析组患者血浆vWF含量与血肌酐之间进行直线相关分析.结果对照组血浆vWF含量为193.65%±39.39%.慢性肾功能不全透析组患者血浆vWF含量为262.18%±59.75%;非透析组患者的vWF含量为224.10%±55.92%,两组均显著高于对照组(P《0.05,P《0.001),而且透析组血浆vWF含量明显高于非透析组(P《0.05).非透析组血浆vWF含量与血肌酐之间无线性关系(r=0.213 4,P》0.05);透析组血浆vWF含量与透析月数之间也无线性关系(r=0.185 2,P》0.05).结论慢性肾功能不全患者存在血管内皮系统损害,从而导致血浆vWF含量升高;血液透析可进一步加重其损害.     

紫癜性肾炎患者血浆TM、vWF和IL-8水平的变化

目的探讨紫癜性肾炎患儿血浆血栓调节蛋白(TM)、血管性假性血友病因子(vWF)和白细胞介素8(IL-8)水平的变化及其与紫癜性肾炎临床分型的相关性。方法选择过敏性紫癜患儿65例(HSP组);其中过敏性紫癜性肾炎(HSPN)51例,无肾损害者14例(HSP单纯型)。51例HSPN患儿分为:孤立性血尿或孤立性蛋白尿18例(HSPN 1型),血尿和蛋白尿14例(HSPN 2型),急性肾炎型11例(HSPN 3型),肾病综合征型8例(HSPN 4型)。对照组为我科门诊体检的健康儿童19例。分别测定血浆TM、vWF、IL-8水平,分析血浆TM、vWF、IL-8与紫癜性肾炎临床分型的关系。结果 HSP组血浆TM、vWF、IL-8高于对照组(P<0.05),HSPN 2、3、4型患儿血浆TM、vWF、IL-8明显高于单纯型(P<0.05)。结论紫癜性肾炎患儿血浆TM、vWF、IL-8水平随着临床分型不同而变化。联合测定有助于紫癜性肾炎的早期诊断和治疗。     

Therapeutic approaches to acquired von Willebrand syndrome

Acquired von Willebrand syndrome (AVWS) is a rare acquired bleeding disorder similar to the congenital von Willebrand disease (VWD) in terms of laboratory findings. Diagnosis of AVWS can be very difficult, with treatment normally taking an empirical form. Although more than 200 cases have been reported since 1968, no retrospective or prospective studies are available on AVWS. Recently, an International Registry on AVWS, gathering data directly from worldwide Departments of Haematology-Oncology and Haemophilia Centres, has been organised by a group working on behalf of the Subcommittee on VWF in the Scientific Standardisation Committee (SSC) of International Society on Thrombosis and Haemostasis (ISTH). Information about an additional 211 AVWS patients is now available, with more detailed data on demography, type of haemorrhage, diagnostic tests for AVWS and management of bleeding episodes. The additional 211 AVWS cases are associated with lymphoproliferative (47%) or myeloproliferative (19%) disorders, cardiovascular diseases, neoplasia (7%) and other miscellaneous diseases (14%). Bleeding episodes of AVWS patients were managed by differnent compounds including desmopressin (22%), FVIII/VWF concentrates (26%) and high-dose immunoglobulin (10%), plasmapheresis (2%), steroids (5%) and immunosuppressive drugs (20%). Based on complied data, we can conclude that none of the therapeutic approaches proposed are 100% effective in all AVWS cases. Therefore, treatment must be customised for each patient according to the underlying disorder, as well as to the type and the severity of bleeding episode and must be targeted to each specific case.     

丹蛭降糖胶囊对2型糖尿病患者血浆血管性假血友病因子及血浆内皮素的影响

目的观察丹蛭降糖胶囊对2型糖尿病患者血浆血管性血友病因子（vWF）和血浆内皮素（ET）的影响。方法64例2型糖尿病患者随机分为2组,单用马来酸罗格列酮者为对照组,合用丹蛭降糖胶囊者为治疗组。治疗2个月,观察两组患者治疗前后vWF、ET的变化。结果丹蛭降糖胶囊治疗组患者治疗后vWF、ET水平显著下调,与治疗前及对照组相比有显著性差异（P〈0．01）。结论丹蛭降糖胶囊通过对vWF和ET的影响,有效地保护血管内皮功能受损,对防治糖尿病血管并发症的发生和发展具有重要意义。     

血管性血友病因子对冠心病发生影响的研究进展

von Willebrand因子又称血管性血友病因子,通过特异的血小板膜受体(糖蛋白Ιb-IX复合物)介导血小板与内皮下层的黏附,是一个反应内皮损伤的标志物,其增高有利于血小板的黏附。vWF与冠心病的发生相关,是反映内皮细胞受损的可靠指标之一。本研究通过对vWF生物学特点、生理功能、作用机理及对冠心病发生影响的相关论文的研究整理,为临床探讨冠心病影响因素及治疗提供参考依据。     

心力衰竭患者血浆纤维结合蛋白、抗凝血酶Ⅲ及Ⅷ因子相关抗原的测定及意义

用免疫电泳法测定心力衰竭(心衰)患者血浆中纤维结合蛋白(Fn),抗凝血酶Ⅲ(AT-Ⅲ及Ⅷ因子相关抗原(vWF)水平。发现Fn在心衰患者显著降低,且随着心衰的病程进展而降低更明显;AT-Ⅲ在心衰患者中显著降低,而在心功能Ⅳ级者中降低最为明显;vWF在心衰患者中显著增高,且随病程而增高更明显。提示Fn和AT-Ⅲ的降低和vWF的增高,可作为评估心衰演变的指标。     

氯沙坦治疗老年高血压病及对内皮素与血管性血友病因子的影响

目的 评价氯沙坦（Ｌｏｓａｒｔａｎ,Ｌｏｓ）治疗老年人轻、中度高血压病的临床疗效及对内皮素－１（ＥＴ－１）和血管性血友病因子（ｖｏｎ ｗｉｌｌｅｂｒａｎｄ ｆａｃｔｏｒ,ｖＷＦ）的影响。方法 选取轻、中度高血压患者６３例,一组了Ｌｏｓ（３３例）５０ｍｇ／ｄ,另一组服Ｂｅｎａｚｅｐｒｉｌ（Ｂｅｎ）（３０例）１０ｍｇ／ｄ,于治疗前及治疗２、４、６、８周末测偶测血压,于治疗前及治疗８周末行动态血压检测（     

阿托伐他汀对充血性心力衰竭患者心功能C反应蛋白和vonWillebrand因子的影响

目的探讨充血性心力衰竭（CHF）患者应用阿托伐他汀后对心功能和C反应蛋白（CRP）、von—Willebrand因子（vwF）的影响及意义。方法随机选择91例CHF患者，EF〈0.35，46例加用阿托伐他汀，另外45例作为对照组。观察治疗前后心功能和CRP、vWF的变化。结果（1）用药前两组患者心功能和CRP、vWF水平差异无统计学意义（P〉0．05）；（2）加用阿托伐他汀的患者，心功能较对照组比较有明显改善（P〈0．05）；（3）用药后两组患者CRP、vWF水平均有明显降低（P〈0．05），但外周血管阻力（SVR）无明显差异；（4）加用阿托伐他汀组的患者，其心率（HR）、CRP、vWF水平均较对照组治疗后有明显降低（P〈0．05）。结论CHF患者加用阿托伐他汀可明显改善心功能，降低HR、血清CRP和vWF水平。     

急性脑梗死患者血小板聚集功能、血管性血友病因子、抗凝血酶及 D-二聚体测定的临床意义

目的：探讨急性脑梗死患者血小板聚集功能（ PAgT）、血管性血友病因子（ vWF）、抗凝血酶（ AT）和D-二聚体（ D-dimer）水平变化及临床意义。方法选用相应的方法和仪器测定112例脑梗死及80例健康对照者血（浆） PAgT、vWF、AT和D-dimer水平变化,同时对部分患者进行治疗前、后的对比分析。结果脑梗死患者血中PAgT、vWF、D-dimer等指标均明显高于健康对照组,AT活性较对照组显著降低,差异有统计学意义（P＜0．05或P＜0．01）。选取经治疗效果明显好转的78例脑梗死患者,出院前取空腹静脉血测定PAgT、vWF、AT、D-dimer等指标,并与治疗前对照,结果治疗后PAgT、vWF、D-dimer降低,AT活性升高,差异有统计学意义（P＜0．05或P＜0．01）。结论脑梗死患者体内存在明显的凝血及纤溶功能异常,与血管内皮损伤、血小板聚集功能增强、凝血及纤溶功能亢进、抗凝功能降低等多因素有关。 PAgT、vWF、AT、D-dimer可以作为脑梗死患者诊断、治疗监测和预后判断的参考指标。     

Bitiscetin-3, a Novel C-Type Lectin-like Protein Cloned from the Venom Gland of the Viper Bitis arietans,Induces Platelet Agglutination and Inhibits Binding of Von Willebrand Factor to Collagen

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and—at least bitiscetin-1—induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein—bitiscetin-3—were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and β share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3β were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit—which differs from that of both bitiscetin-3 subunits—the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and β-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.     

冠心病患者血液中肺炎衣原体抗体、血管性假性血友病因子和血脂的变化及其临床意义

目的研究冠心病（CHD）患者肺炎衣原体（Cpn）感染与血管性假性血友病因子（vWF）、血脂的相互关系,从而探讨CHD可能的发病机制。方法将CHD患者120例分为急性心肌梗死（AMI）组44例、不稳定心绞痛（UAP）组36例、稳定性心绞痛（SAP）组40例,另选取60例正常人作为对照组。所有对象均通过微量免疫荧光法测量Cpn特异性抗体,用酶联吸附法测定vWF,用全自动生化仪测定血脂。结果AMI组、UAP组Cpn特异性抗体阳性率高于对照组,差异有统计学意义（P〈0．05）。Cpn特异性抗体阳性患者vWF及血清总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白胆固醇（LDL-c）水平高于Cpn特异性抗体阴性者,高密度脂蛋白胆固醇（HDL—C）水平低于对照组,差异有统计学意义（P〈0．05）;且血浆vWF及TC、TG、LDL-C水平与Cpn特异性培G抗体几何平均滴度呈正相关,HDL—C水平与之呈负相关。结论Cpn致CHD的可能机制为：Cpn感染使体内vWF水平升高,协同血脂异常,激发和加重冠状动脉内炎性反应,促进患者血液黏稠度增加,动脉血栓发生率升高,参与动脉粥样硬化（AS）的发生发展。     

Von Willebrand factor and fibrinolytic parameters during the desmopressin test in patients with Cushing's disease

AIMS

Desmopressin, a vasopressin analogue, is used for various clinical purposes, including haemostasis and, in recent times, the diagnostic work-up of patients with Cushing''s syndrome, a condition associated with a known prothrombotic profile. We decided to evaluate whether and to what extent a diagnostic dose of desmopressin induces significant changes in endothelial parameters in patients with Cushing''s disease (CD) and obese and normal weight controls.

METHODS

Twelve patients with CD, 10 obese and five normal weight controls were studied. Von Willebrand antigen (VWF : Ag), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) were measured at baseline and up to 4 h after 10 µg desmopressin i.v.

RESULTS

Desmopressin 10 µg transiently increased VWF : Ag and t-PA and decreased PAI-1 in all subjects. The magnitude of the VWF : Ag and t-PA increases after desmopressin was comparable in the three groups (VWF : Ag peak-to-basal ratio 1.9 ± 0.17, 1.5 ± 0.11 and 1.8 ± 0.13 and t-PA peak-to-basal ratio 1.6 ± 0.18, 1.6 ± 0.20 and 1.8 ± 0.24 for CD, obese and controls, respectively, all NS). The PAI-1 decrease observed in patients with CD was comparable with obese (0.7 ± 0.07 and 0.6 ± 0.09, NS) and controls (0.7 ± 0.07 vs. 0.4 ± 0.09, P = 0.08).

CONCLUSIONS

Administration of desmopressin to patients with CD for diagnostic purposes induces a transitory increase in VWF : Ag counterbalanced by a decrease in PAI-1 and increase in t-PA. The magnitude of these changes is largely comparable with that observed in obese and normal weight controls. Our data show that testing with desmopressin does not induce disease-specific changes in endothelial markers in patients with CD.     

慢性肾病患者血浆vWF与vWF裂解酶的检测及临床意义

目的 探讨慢性肾脏病(CKD)患者血浆血管性血友病因子(vWF)水平及vWF裂解酶(vWF-CP)活性检测及其在CKD中的临床意义.方法 31例狼疮肾炎(LN组)、45例糖尿病.肾病(DN组)、25例原发性肾病综合征(NS组)、38例慢性肾炎(CGN组)患者及40例正常对照者(NC组),采用残余胶原结合实验法及ELlSA法检测血浆vWF-CP活性及vWF水平,并分析不同病理类型、不同肾功能状态下两项指标的改变.结果 4组CKD患者血浆vWF水平均显著高于NC组(P<0.01),vWF-CP活性显著低于NC组(P<0.01);其中,LN、NS组下降最明显;CKD患者vWF含量与vWF-CP活性显著负相关.CKDⅡ、Ⅲ期vWF-CP活性显著低于Ⅰ、Ⅳ、V期(P<0.05).行肾活检患者中,LN患者Ⅳ型vWF显著高于Ⅱ型、Ⅲ型,而vWF-CP活性显著低于Ⅱ型、Ⅲ型.膜性肾炎(MN)vWF-CP活性显著低于微小病变肾病(MCD)、系膜增生性肾炎(MsPGN)和IgA肾病(IgAGN)(P<0.05).经综合治疗8周后,vWF水平显著下降,vWF-CP活性水平在治疗后SLEDAJ评分<9分者有显著性升高(P<0.05).结论 CKD患者血浆vWF升高,vWF-CP活性低下程度与肾病的病种有关,反映了病理类型的轻莺.     

Structure and function of snake venom cysteine-rich secretory proteins.

Cysteine-rich secretory proteins (CRISPs) are primarily found in the epididymis of mammals and are expressed in diverse organisms. However, the functions of most CRISPs remain unknown. Recent studies reveal that CRISPs are widely distributed in snake venoms and that they inhibit smooth muscle contraction and cyclic nucleotide-gated ion channels. In this review, we discuss recent findings on several snake venom-derived CRISPs.     

The Perioperative von Willebrand Factor Activity and Factor VIII Levels Among Alcohol Use Disorder Patients Undergoing Total Knee or Hip Replacement

Alcohol use disorder patients have a five-fold higher risk of postoperative bleeding complications. We measured the perioperative von Willebrand factor and factor VIII levels in consecutive patients with alcohol use disorder. In one university hospital, 105 patients scheduled for arthroplasty were screened, and 25 fulfilled inclusion criteria. Postoperatively, we found significantly decreased von Willebrand factor ristocetin cofactor values over time among alcohol use disorder patients and significantly different time courses of factor VIII levels between patients with and without a diagnosed alcohol use disorder. Blood loss was significantly increased among alcohol use disorder patients on their first postoperative day.     

血浆白蛋白、血管性血友病因子检测在脑卒中监测中的作用

目的 探讨白蛋白（ALB）和血管性血友病因子（vWF）在脑卒中疾病中的改变。方法 对145例脑卒中患者（恢复良好组95例、恢复不良组50例）在初发2周内和6个月时进行ALB和vWF检测，并观察其和疾病恢复的关系。结果 恢复良好组（mRS≤2）ALB浓度明显高于恢复不良组（mRS≥3）（P〈0．05），且两组在发病6个月后的ALB浓度更低。另外，脑卒中患者的vWF浓度显著高于正常对照，且发病2周内显著高于发病6个月后（P〈0．05）。恢复不良组虽然高于恢复良好组，但在统计学上没有明显差别。结论 ALB和vWF在脑卒中患者的检测有助于了解疾病的活跃程度和对预后的评估。