A Novel Family of Anticoagulants from Ixodes scapularis

描述（由申请人提供）：本提案的总体目标是评估一种新型蜱类唾液蛋白家族的抗凝血特性，黑腿蜱，当它吞噬脊椎动物宿主时绕过宿主的止血机制。这种蜱虫的唾液含有有效的抗凝血剂，包括Xa因子抑制剂。对具有抗凝活性的纯化唾液蛋白进行n端测序。克隆了相应的蜱唾液cDNA，编码一个96kda的蛋白，命名为Salp9A。该蛋白与Salp14同源，我们之前通过免疫筛选蜱唾液腺cDNA表达文库鉴定了Salp14。额外的蛋白质和cDNA测序表明，I肩胛骨唾液腺表达至少10种不同的salp14相关蛋白。我们表达并纯化了重组Salp14，发现其抑制凝血和Xa因子。这些数据表明我们发现了一个新的抗凝血剂家族。在I期实验中，我们将获得编码选定Salp14家族成员的全长cdna。重组蛋白将由这些cdna表达。重组蛋白将在抗凝血和蛋白酶抑制试验中与部分纯化的唾液抗凝血剂进行比较。这些研究将涉及结构和功能，并为选择抗凝血分子进行II期动物实验提供合理依据。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to evaluate the anticoagulant properties of a family of novel tick salivary proteins Ixodes scapularis, the black-legged tick, circumvents host hemostatic mechanisms as it engorges on vertebrate hosts. This tick's saliva contains potent anticoagulants, including a Factor Xa inhibitor. Purified salivary proteins with anticoagulant activity were subjected to N-terminal sequencing. A corresponding tick salivary cDNA was cloned that encoded a 96 kDa protein, designated Salp9A. This protein is homologous to Salp14, which we previously identified by immunoscreening a tick salivary gland cDNA expression library. Additional protein and cDNA sequencing indicate that I scapularis salivary glands express at least ten different Salp14-related proteins. We expressed and purified recombinant Salp14, and found that it inhibits coagulation and Factor Xa. These data suggest that we have discovered a new family of anticoagulants. In Phase I experiments we will obtain full-length cDNAs encoding selected Salp14 family members. Recombinant proteins will be expressed from these cDNAs. The recombinant proteins will be compared to partially purified salivary anticoagulants in anticoagulant and protease inhibition assays. These studies will relate structure and function, and provide a rational basis for selecting anticoagulant molecules for Phase II animal studies.