High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalization and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. Therefore, we investigated the binding and subsequent distribution of biotinylated-HK (biotin-HK) associated with human umbilical vein endothelial cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avidity at 1 to 3 hours at 37 degrees C than at 4 degrees C (Bmax = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v Bmax = 2.6 +/- 0.2 x 10(6) molecules/cell, kd = 46 +/- 8 nmol/L). However, there was no evidence that the difference was caused by internalization of HK at the higher temperature. First, the same amount of biotin-HK was associated with nonpermeabilized and permeabilized HUVEC using buffers containing 20 to 50 mumol/L zinc ion in the absence or presence of 2 mmol/L calcium ion. Second, binding of biotin-HK to HUVEC was approximately 92% reversible at 1 hour when the cells were maintained at both 37 degrees C and 4 degrees C. Third, neither chloroquine nor primaquine altered the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeable dye, crystal violet, almost completely quenched the fluorescence signal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to the membrane as shown by laser scanning confocal microscopy. The expression of HK binding sites had an absolute requirement for metabolic energy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocking human alpha-thrombin binding and its resultant induction of prostacyclin formation. These studies indicate that HK is not internalized by HUVEC, but remains primarily on cell surfaces to be accessible for BK liberation and to modulate the binding and actions of alpha-thrombin.     

High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.     

A prospective international cooperative information technology platform built using open-source tools for improving the access to and safety of bone marrow transplantation in low- and middle-income countries

Jagriti Innovations developed a collaboration tool in partnership with the Cure2Children Foundation that has been used by health professionals in Italy, Pakistan, and India for the collaborative management of patients undergoing bone marrow transplantation (BMT) for thalassemia major since August 2008. This online open-access database covers data recording, analyzing, and reporting besides enabling knowledge exchange, telemedicine, capacity building, and quality assurance. As of February 2014, over 2400 patients have been registered and 112 BMTs have been performed with outcomes comparable to international standards, but at a fraction of the cost. This approach avoids medical emigration and contributes to local healthcare strengthening and competitiveness. This paper presents the experience and clinical outcomes associated with the use of this platform built using open-source tools and focusing on a locally pertinent tertiary care procedure—BMT.     

Takayasu arteritis complicating pregnancy in adolescence

Pregnant adolescent patients afflicted with Takayasu arteritis represent a clinical entity not seen by many. The care of such patients is often managed by multidisciplinary teams, where vascular surgeons are asked to provide input on cardiovascular implications during and after a pregnant state. Knowledge and understanding of the interaction between the two conditions allows for well-informed decision making and favorable outcomes with pregnancy, as well as proper long-term follow-up and care with appropriate clinicians.     

Biophysical characteristics of skin in diabetes: a controlled study

Background  Cutaneous complications are common in diabetes. Previous assays suggest that hyperglycemia and decreased insulin signal are involved in the impairment of skin function. The aim of this study was to evaluate the biophysical characteristics of skin in patients with diabetes mellitus and compares them with healthy non-diabetic controls.
Objective  To measure biophysical characteristic of skin including transepidermal water loss (TEWL), water content, sebum and skin elasticity in patients with diabetes mellitus and compare them with healthy non-diabetic controls.
Methods  This case-control study was conducted on 38 patients with diabetes and 40 age- and sex-matched healthy people. The biophysical properties of skin including stratum corneum (SC) hydration, sebum content, TEWL and skin elasticity were measured and compared between the two groups at three different locations of the body.
Results  The measurement of SC hydration and TEWL showed no significant difference between diabetics and controls. The skin surface lipids on the forehead but not other sites were significantly lower in the diabetics than in the controls. Acoustic wave propagation speed, a measurement related to skin elasticity, was significantly lower in forearm and forehead of diabetics.
Conclusion  Diabetes affects some functional properties of epidermis and dermis that may responsible for many cutaneous manifestations of diabetes. These results suggest that patients with diabetes mellitus tend to show a normal hydration state of the SC together with decreased sebaceous gland activity and impaired skin elasticity, without any impairment of the SC barrier function.

Conflicts of interest

None declared