PubMed · 10137586
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I C McManus. 1994. Knowing the knowledge.. https://doi.org/10.1136/qshc.3.2.67
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Thrombin acts as a procoagulant when it cleaves fibrinogen and promotes the formation of a fibrin clot and functions as an anticoagulant when it activates protein C with the assistance of the cofactor thrombomodulin. The dual function of thrombin in the blood poses the challenge to turn the enzyme into a potent anticoagulant by selectively abrogating fibrinogen cleavage. Using functional and structural data, we have rationally designed a thrombin mutant, W215A/E217A, that cleaves fibrinogen with a value of k(cat)/K(m) about 20,000-fold slower than wild-type but activates protein C in the presence of thrombomodulin with a specificity comparable with wild-type. This mutant demonstrates for the first time that the relative specificity of thrombin toward fibrinogen and protein C can be completely reversed.
We have examined whether oxidized low-density lipoprotein (ox-LDL) affects the function of tissue-factor-pathway inhibitor (TFPI), an anti-coagulant regulator in the extrinsic pathway of coagulation, in cultured human umbilical vein endothelial cells (HUVEC). Treatment of culture medium of HUVEC with ox-LDL, but not with native or acetylated LDLs, drastically decreased the reactivity of TFPI to its antibody specific for Kunitz domain 1 or one specific for the conformation between Kunitz 1 and 2 of TFPI, and caused a rapid, concentration-dependent decrease in the functional activity of TFPI to inhibit Factor X activation. When 5 ng of recombinant TFPI (rTFPI) was mixed with 10 microg of ox-LDL for 30 min, almost all of the rTFPI was detected in the ox-LDL fraction and no free rTFPI was observed on non-denaturing PAGE, in contrast with the virtual absence of rTFPI in the native LDL fraction. Ox-LDL decreased the antigen level of TFPI in the lysate of HUVEC in a time-dependent manner. It did not affect the mRNA level, but ox-LDL-dependent reduction of the TFPI antigen level in HUVEC was reversed by the simultaneous treatment of ox-LDL with bafilomycin A1, an inhibitor of the lysosomal proton pump. These results indicate that ox-LDL lessens the anti-coagulant function of TFPI through both oxidative modification and accelerated degradation of the molecule outside and inside HUVEC respectively.
Primary pulmonary hypertension (PPH) is a syndrome of dyspnea, chest pain, and syncope defined by increased pulmonary vascular resistance and the absence of a known cause. It also occurs in a familial form, which is linked to unidentified genes on chromosome 2. This syndrome is characterized by abnormalities of pulmonary vascular biology in each compartment of the blood vessel. The lumen has a prothrombotic diathesis, the endothelium displays an excessive production of vasoconstrictors relative to vasodilators, and the smooth muscle cells are depolarized and calcium-overloaded, which is due in part to reduced expression of voltage-gated potassium channels (Kv). This causes vasoconstriction and may promote cell proliferation. The adventitia displays excessive remodeling, which is associated with exaggerated metalloproteinase and elastase activity. Conceptually, PPH seems to require a permissive genotype, a susceptible phenotype (eg, endothelial dysfunction) and, in many cases, an exogenous trigger (eg, an anorexigen). Although there is not a generally accepted, unifying hypothesis regarding its cause, impaired function and the expression of vascular and platelet Kv channels suggest PPH may be a disease of the ion channels. Abnormal matrix metalloproteinase and elastase activity could also explain the abnormal vascular tone, platelet activation, and remodeling in PPH. Although calcium-channel blockers and prostacyclin, particularly when coadministered with warfarin, improve survival, PPH has a 5-year mortality rate of approximately 50%. Pharmacological and gene therapies aimed at enhancing the activity of prostacyclin, nitric oxide synthases, and Kv channels or at inhibiting endothelin and matrix metalloproteinases are promising areas for future development.