Diagnostic characteristics of the 20-minute whole blood clotting test in detecting venom-induced consumptive coagulopathy following carpet viper envenoming

Introduction Envenoming by Echis spp. (carpet or saw-scaled vipers) causes haemorrhage and coagulopathy and represents a significant proportion of snakebites in the savannah regions of West Africa. Early diagnosis of envenoming is crucial in the management of these patients and there is limited evidence on the utility of the 20-minute whole blood clotting test (20WBCT) in diagnosing venom-induced consumptive coagulopathy (VICC) following envenoming by Echis ocellatus. Methods A prospective observational cohort study was conducted at the Kaltungo General Hospital in North-eastern Nigeria from September 2019 to September 2021. Standardised 20WBCTs were conducted by trained hospital staff and citrated plasma samples were collected at numerous timepoints. Prothrombin time (PT) and international normalised ratio (INR) were determined using a semi-automated analyser and INR values were calculated using international sensitivity indices (ISI). The sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and likelihood ratios of the 20WBCT compared to an INR ≥ 1.4 were calculated, alongside 95% confidence intervals. Results We enrolled 121 patients into our study, with a median age of 26 (18.0–35.0) years and a male predominance (75.2%). The 20WBCT was positive (abnormal) in 101 out of 121 patients at timepoint 0h, of which 95 had an INR ≥ 1.4, giving a sensitivity of 87.2% (95%CI 79.4–92.8). Among patients with a negative 20WBCT (normal), six had an INR < 1.4 giving a specificity of 50% (95%CI 21.1–78.9%). The positive and negative likelihood ratios were 1.7 (95%CI 1.6–1.9) and 0.3 (95%CI 0.1–0.4) respectively. Conclusion The 20WBCT is a simple, cheap, and easily accessible bedside test with a high sensitivity for the detection of patients with venom induced consumptive coagulopathy (VICC) following envenoming by E. ocellatus, although false positives do occur. Repeated 20WBCTs can identify patients with new, persistent, and rebound coagulopathy

A key role for tetrahydrobiopterin-dependent endothelial NOS regulation in vascular resistance arteries: studies in endothelial cell tetrahydrobiopterin-deficient mice

Background and purpose: The cofactor tetrahydrobiopterin (BH4) is a critical regulator of endothelial NOS (eNOS) function, eNOS-derived NO and reactive oxygen species (ROS) signalling in vascular physiology. To determine the physiological requirement for de-novo endothelial cell BH4 synthesis in vasomotor function in resistance arteries, we have generated a mouse model with endothelial cell-specific deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme for BH4 biosynthesis, and evaluated BH4-dependent eNOS regulation, eNOS-derived NO and ROS generation. Experimental approach: Wire myography was used to assess the reactivity of mouse 2nd order mesenteric arteries. High-performance liquid chromatography was used to determine BH4, BH2 and biopterin. Western blotting was used for expression analysis. Key Results: Gch1fl/flTie2cre mice demonstrated reduced GTPCH protein and BH4 levels in mesenteric arteries. Deficiency in endothelial cell BH4 leads to eNOS uncoupling, increased ROS production and loss of NO generation in mesenteric arteries of Gch1fl/flTie2cre mice. Gch1fl/flTie2cre mesenteric arteries had enhanced vasoconstriction to U46619 and phenylephrine, which was equalised by L-NAME. Endothelium-dependent vasodilatations to ACh and SLIGRL were impaired in mesenteric arteries from Gch1fl/flTie2cre mice compared to wild-type littermates. The loss of eNOS-derived NO-mediated vasodilatation was associated with increased eNOS-derived H2O2 and prostaglandin-derived vasodilator in Gch1fl/flTie2cre mesenteric arteries. Conclusions and implications: Endothelial cell Gch1 and BH4-dependent eNOS regulation play pivotal roles in maintaining vascular homeostasis in resistance arteries. Therefore, targeting vascular Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of microvascular dysfunction in patients with cardiovascular disease

Endothelial cell tetrahydrobiopterin modulates sensitivity to Ang (Angiotensin) II–induced vascular remodeling, blood pressure, and abdominal aortic aneurysm

GTP cyclohydrolase 1 (GTPCH, encoded by Gch1) is required for the synthesis of tetrahydrobiopterin (BH4); a critical regulator of endothelial NOS (eNOS) function. We have previously shown that mice with selective loss of Gch1 in endothelial cells have mild vascular dysfunction, but the consequences of endothelial cell BH4 deficiency in vascular disease pathogenesis is unknown. We investigated the pathological consequence of angiotensin II (Ang II) infusion in endothelial cell Gch1 deficient (Gch1fl/flTie2cre) mice. Ang II (0.4 mg/kg/day, delivered by osmotic mini pump) caused a significant decrease in circulating BH4 levels in Gch1fl/flTie2cre mice and a significant increase in the L-NAME inhabitable production of H2O2 in the aorta. Chronic treatment with this subpressor dose of Ang II resulted in a significant increase in blood pressure only in Gch1fl/flTie2cre mice. This finding was mirrored with acute administration of Ang II where increased sensitivity to Ang II was observed at both pressor and subpressor doses. Chronic Ang II infusion in Gch1fl/flTie2ce mice resulted in vascular dysfunction in resistance mesenteric arteries with an enhanced constrictor and decreased dilator response, and medial hypertrophy. Altered vascular remodelling was also observed in the aorta with an increase in the incidence of abdominal aortic aneurysm (AAA) formation in Gch1fl/flTie2ce mice. These findings indicate a specific requirement for endothelial cell BH4 in modulating the hemodynamic and structural changes induced by Ang II, through modulation of blood pressure, structural changes in resistance vessels, and aneurysm formation in the aorta

Endothelial cell tetrahydrobiopterin modulates sensitivity to Ang (Angiotensin) II–induced vascular remodeling, blood pressure, and abdominal aortic aneurysm

GTP cyclohydrolase 1 (GTPCH, encoded by Gch1) is required for the synthesis of tetrahydrobiopterin (BH4); a critical regulator of endothelial NOS (eNOS) function. We have previously shown that mice with selective loss of Gch1 in endothelial cells have mild vascular dysfunction, but the consequences of endothelial cell BH4 deficiency in vascular disease pathogenesis is unknown. We investigated the pathological consequence of angiotensin II (Ang II) infusion in endothelial cell Gch1 deficient (Gch1fl/flTie2cre) mice. Ang II (0.4 mg/kg/day, delivered by osmotic mini pump) caused a significant decrease in circulating BH4 levels in Gch1fl/flTie2cre mice and a significant increase in the L-NAME inhabitable production of H2O2 in the aorta. Chronic treatment with this subpressor dose of Ang II resulted in a significant increase in blood pressure only in Gch1fl/flTie2cre mice. This finding was mirrored with acute administration of Ang II where increased sensitivity to Ang II was observed at both pressor and subpressor doses. Chronic Ang II infusion in Gch1fl/flTie2ce mice resulted in vascular dysfunction in resistance mesenteric arteries with an enhanced constrictor and decreased dilator response, and medial hypertrophy. Altered vascular remodelling was also observed in the aorta with an increase in the incidence of abdominal aortic aneurysm (AAA) formation in Gch1fl/flTie2ce mice. These findings indicate a specific requirement for endothelial cell BH4 in modulating the hemodynamic and structural changes induced by Ang II, through modulation of blood pressure, structural changes in resistance vessels, and aneurysm formation in the aorta