Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA.
Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, WI, USA.
J Thromb Haemost. 2019 Jan;17(1):63-71. doi: 10.1111/jth.14341. Epub 2018 Dec 18.
Essentials Defective binding to collagen IV has been seen in von Willebrand factor (VWF) A1 domain variants. We developed a murine model of defective VWF-collagen IV interactions with VWF variant p.R1399H. p.1399HH homozygous mice had decreased binding to collagen IV and increased bleeding times. p.1399HH homozygous mice had increased time to thrombosis and decreased platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) binding to type IV collagen occurs via the VWF A1 domain, with p.R1399H being the most common VWF variant affecting this interaction. Objectives We generated a murine model of 1399H VWF to investigate its in vivo effects. Methods Mice expressing the murine 1399H variant were generated via gene targeting in embryonic stem cells. VWF antigen and VWF collagen binding were measured with ELISA. Tail bleeding time assays were performed by clipping a 3-mm segment. Ferric chloride-induced thrombosis was measured via ultrasound in the carotid artery. Platelet aggregation in response to collagens I and IV was measured. VWF-dependent platelet adhesion to collagen IV was measured under flow. Results Breeding of heterozygous p.R1399H and homozygous p.1399HH mice was observed to follow normal Mendelian ratios. No spontaneous bleeding was observed for any of the offspring. VWF expression was normal, but VWF binding to collagen IV was decreased in both heterozygous and homozygous offspring. Blood loss following tail resection was increased for p.1399HH mice, and occlusion times following ferric chloride-induced thrombosis were prolonged. Platelet aggregation was unaffected, but platelet adhesion to collagen IV under flow was diminished for p.1399HH mice. Conclusions These results show that a decrease in the ability of 1399H VWF to bind collagen IV under static conditions corresponds to a decrease in binding under flow conditions, an increased bleeding time, and a prolonged time to thrombosis. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagen IV binding.
主要缺陷 已在血管性血友病因子 (VWF) A1 结构域变异体中观察到与胶原 IV 的结合缺陷。我们开发了一种 VWF 与变异型 p.R1399H 相互作用缺陷的小鼠模型。p.1399HH 纯合子小鼠与胶原 IV 的结合减少，出血时间延长。p.1399HH 纯合子小鼠血栓形成时间延长，血小板黏附减少。
背景 血管性血友病因子 (VWF) 与 IV 型胶原的结合发生在 VWF A1 结构域，p.R1399H 是最常见的影响这种相互作用的 VWF 变异体。
目的 我们生成了一种 1399H VWF 的小鼠模型，以研究其体内效应。
方法 通过胚胎干细胞中的基因靶向生成表达小鼠 1399H 变异体的小鼠。通过 ELISA 测量 VWF 抗原和 VWF 胶原结合。通过剪断 3mm 段进行尾部出血时间测定。通过颈动脉内氯化铁诱导的血栓形成的超声测量评估血栓形成。通过胶原 I 和 IV 诱导的血小板聚集测量评估血小板聚集。在流动条件下测量 VWF 依赖性血小板黏附至胶原 IV。
结果 观察到 p.R1399H 杂合子和 p.1399HH 纯合子小鼠的繁殖符合正常孟德尔比例。没有观察到任何后代发生自发性出血。VWF 表达正常，但杂合子和纯合子后代的 VWF 与胶原 IV 的结合均减少。p.1399HH 小鼠的尾部切除后失血量增加，氯化铁诱导的血栓形成后的闭塞时间延长。血小板聚集不受影响，但 p.1399HH 小鼠的血小板在流动条件下黏附至胶原 IV 的能力降低。
结论 这些结果表明，1399H VWF 在静态条件下结合胶原 IV 的能力下降，与在流动条件下的结合能力下降、出血时间延长和血栓形成时间延长相对应。这项研究支持 VWF 与胶原 IV 结合异常的患者存在出血表型的可能性。
