Enzymes produced by autoactivation of blood factor XII in buffer: A contribution from the Hematology at Biomaterial Interfaces Research Group

High-resolution electrophoresis of FXII-derived proteins produced by contact activation of FXII in buffer solutions (i.e. in absence of plasma proteins) with hydrophilic and silanized-glass activators spanning the observable range of water wettability (hydrophilic to hydrophobic), shows no evidence of proteolytic cleavage of FXII into αFXIIa or βFXIIa. The autoactivation mixture contains only a single-chain protein with a molecular weight of ∼80 kDa, confirming Oscar Ratnoff's previous finding of a single-chain activated form of FXII that he called ‘HF_ea’. Functional assays have shown that these autoactivation products exhibit procoagulant potential (protease activity inducing clotting of blood) or amidolytic potential (cleaves amino bonds in s-2302 chromogen but do not cause coagulation of plasma) or both amidolytic potential and procoagulant potential. Some of these proteins also have the remarkable potential to ‘suppress autoactivation’ (i.e. suppress creation of enzymes with procoagulant potential). It is thus hypothesized that autoactivation of FXII in the absence of plasma proteins generates not just a single type of activated conformer, as suggested by previous researchers, but rather an ensemble of conformer products with collective activity that varies with activator surface energy used in contact activation of FXII. Furthermore, reaction of αFXIIa with FXII in buffer solution does not produce additional αFXIIa by the putative autoamplification reaction FXIIa + FXII → 2FXIIa as has been proposed in past literature to account for the discrepancy between chromogenic and plasma-coagulation assays for αFXIIa in buffer solution. Instead, net procoagulant activity measured directly by plasma-coagulation assays, decreases systematically with increasing FXII solution concentration. Under the same reaction conditions, chromogenic assay reveals that net amidolytic activity increases with increasing FXII solution concentration. Thus, although autoamplification does not occur it appears that there is some form of “FXII self reaction” that influences products of αFXIIa reaction with FXII. Electrophoretic measurements indicate that no proteolytic cleavage takes in this reaction leading us to conclude that change in activity is most likely due to change(s) in FXII conformation (with related change in enzyme activity).     

Competitive-protein adsorption in contact activation of blood factor XII

Contact activation of blood factor XII (FXII, Hageman factor) is moderated by the protein composition of the fluid phase in which FXII is dissolved. Solution yield of FXIIa arising from FXII contact with hydrophilic activating particles (fully water-wettable glass) suspended in a protein cocktail is shown to be significantly greater than that obtained under corresponding activation conditions in buffer solutions containing only FXII. By contrast, solution yield of FXIIa arising from FXII contact with hydrophobic particles (silanized glass) suspended in protein cocktail is sharply lower than that obtained in buffer. This confirms that contact activation is not specific to anionic hydrophilic surfaces as proposed by the accepted biochemistry of surface activation. Rather, contact activation in the presence of proteins unrelated to the plasma coagulation cascade leads to an apparent specificity for hydrophilic surfaces that is actually due to a relative diminution of activation at hydrophobic surfaces and an enhancement at hydrophilic surfaces. Furthermore, the rate of FXIIa accumulation in whole-plasma and buffer solution is found to decrease with time in the continuous presence of activating surfaces, leading to a steady-state FXIIa yield dependent on the initial FXII solution concentration for both hydrophilic and hydrophobic procoagulant particles suspended in either plasma, protein cocktail, or buffer. These results strongly suggest that activation competes with an autoinhibition reaction in which FXIIa itself inhibits FXII-->FXIIa. Experimental results are modeled using a reaction scheme invoking FXII activation and autoinhibition linked to protein adsorption to procoagulant surfaces, where FXII activation is presumed to proceed by either autoactivation (FXII-->surface-->FXIIa) and autohydrolysis (FXII-->FXIIa-->2FXIIa) in buffer solution or autoactivation and reciprocal activation (kallikrein-mediated hydrolysis) in plasma. FXII adsorption competition with other proteins in the fluid phase is proposed to affect the balance of activation and autoinhibition, leading to the observed moderation of FXIIa yield.     

Contributions of contact activation pathways of coagulation factor XII in plasma

Activation of human blood plasma coagulation by contact with hydrophilic or hydrophobic surfaces (procoagulants) is dominated by kallikrein (Kal)-mediated activation of the blood zymogen FXII (Hageman Factor). Mathematical modeling of prekallikrein (PK)-deficient platelet-poor plasma (d(PK)PPP) and PK-reconstituted d(PK)PPP (Rd(PK)PPP) coagulation shows that autoactivation of FXII (FXII-->[surface]FXII) produces no more than about 25% of the total FXIIa produced by the intrinsic pathway. Autoactivation and reciprocal-activation increase in the same proportion with procoagulant surface energy (water-wettability), whereas total amount of FXIIa produced per-unit-area procoagulant remains roughly constant for any particular procoagulant. These results suggest that procoagulant surfaces initiate the intrinsic cascade by producing a bolus of FXIIa in proportion to surface energy or surface area but play no additional role in subsequent molecular events in the cascade. Results further suggest that reciprocal-activation occurs in proportion to the amount of FXIIa produced by the initiating autoactivation step.     

Amidolytic, procoagulant, and activation-suppressing proteins produced by contact activation of blood factor XII in buffer solution

The relative proportions of enzymes with amidolytic or procoagulant activity produced by contact activation of the blood zymogen factor XII (FXII, Hageman factor) in buffer solution depends on activator surface chemistry/energy. As a consequence, chromogenic assay of amidolytic activity (cleavage of amino acid bonds in s-2302 chromogen) does not correlate with the traditional plasma coagulation time assay for procoagulant activity (protease activity inducing clotting of blood plasma). Amidolytic activity did not vary significantly with activator particle surface energy, herein measured as water adhesion tension τ(o)=γ(lv)(o)cosθ(a) ; where γ(lv)(o) is pure buffer interfacial tension and θ(a) is the advancing contact angle. By contrast, procoagulant activity varied as a parabolic-like function of τ(o), high at both hydrophobic and hydrophilic extremes of activator surface energy and falling through a broad minimum within a 20<τ(o)<40 mJ/m(2) (55°<θ(a) < 75°) range, corroborating and expanding previously-published work. It is inferred from these functional assays that an unknown number of protein fragments are produced by contact activation of FXII (a.k.a. autoactivation) rather than just αFXIIa and βFXIIa as popularly believed. Autoactivation products produced by activator particles within the 20<τ(o)<40 mJ/m(2) (55°<θ(a) < 75°) surface-energy range suppresses production of procoagulant enzymes by activators selected from the hydrophobic or hydrophilic surface-energy extremes through as-yet unknown biophysical chemistry. Suppression proteins may be responsible for the experimentally-observed autoinhibition of the autoactivation reaction.     

Elevated levels of soluble glycoprotein V - The plasma marker of platelet activation by thrombin in patients with early stage primary biliary cholangitis (PBC)

PurposeThere is a growing body of evidence for a prothrombotic tendency in patients with primary biliary cholangitis (PBC). The aim of the study was to evaluate coagulation disorders in patients with early stage PBC compared to healthy controls and evaluation of their relationship with clinical data, with particular emphasis on minimal hepatic encephalopathy (MHE).Patients and methodsFifty-one participants (PBC group – 38 patients, all patients but one Child-Pugh A; control group – 13 healthy controls) were included in our prospective, single center study. We assessed the plasma levels of sGPV, plasma procoagulant phospholipids (PPL) and rotational thromboelastometry (ROTEM) profiles in all study participants. Porto-systemic encephalopathy syndrome test was used to assess MHE.ResultsThe sGPV levels were higher in the PBC group compared to the controls: 36.07 ?± ?11.32 ?ng/mL vs 27.04 ?± ?11.72 ?ng/mL, p ?= ?0.031. The PPL level was lower in the PBC group compared to controls resulting in increased clotting time in a factor Xa-based coagulation assay: 54.65 (47.83–58.83) sec. vs 45.90 (43.3–50.5) sec., p ?= ?0.0065. PPL levels were correlated with platelet count (rho ?= ??0.46, p ?= ?0.001). ROTEM parameters did not differ significantly between groups. Coagulation parameters did not differ significantly between patients with and without MHE.ConclusionsWe have showed increased levels of sGPV - a plasma marker of platelet activation by thrombin in patients with early stage PBC compared to healthy controls. We found no relationship between the coagulation disorders and the occurrence of MHE. The PPL level was lower in the PBC group.     

Autoactivation of blood factor XII at hydrophilic and hydrophobic surfaces

Contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution is shown not to be specific for anionic hydrophilic procoagulants as proposed by the accepted biochemistry of surface activation. Rather, FXII activation in the presence of plasma proteins leads to an apparent specificity for hydrophilic surfaces that is actually due to a relative diminution of the FXII-->FXIIa reaction at hydrophobic surfaces. FXII activation in neat-buffer solution was effectively instantaneous upon contact with either hydrophilic (fully water-wettable clean glass) or hydrophobic (poorly water-wettable silanized glass) procoagulant particles, with greater FXIIa yield obtained by activation with hydrophobic procoagulants. In sharp contrast, both activation rate and yield was found to be significantly attenuated at hydrophobic surfaces in the presence of plasma proteins. Putative FXIIa produced by surface activation with both hydrophilic and hydrophobic procoagulants was shown to hydrolyze blood factor XI (FXI) to the activated form FXIa (FXIFXIIa-->FXIa) that causes FXI-deficient plasma to rapidly coagulate.     

采用MDA-MB-231细胞膜进行表皮生长因子受体活性的检测

目的　探讨能否直接利用肿瘤细胞膜进行表皮生长因子受体 (Epidermalgrowthfactorreceptor,EGFR)催化活性检测。方法　首先筛选出EGFR基因表达水平相对较高的细胞株MDA MB 2 31,通过差速离心制备细胞膜 ,采用Westernblotting检测EGFR催化磷酸化的程度。结果　底物被磷酸化 ,加入特异性拮抗剂AG14 78后 ,磷酸化被抑制。结论　利用肿瘤细胞膜检测EGFR活性的设想是成立的 ,并且其方法简便、经济。     

Additional factor XII (hageman factor) deficiency in hemophilia a and in von willebrand syndrome

Summary Factor XII plasma levels were investigated with several methods in patients with hemophilia A and B and von Willebrand syndrome. There seem to be some families with hemophilia A or von Willebrand syndrome, who have an additional, congenital, partial lack of factor XII (Hageman factor). The mode of inheritance is independent of the other coagulation disorder. Frequently, the first indication of an additional factor XII deficiency is the disproportionate prolongation of the activated partial thromboplastin time (PTT) as regards the factor VIII level. The average factor XII level in patients with hemophilia A and von Willebrand syndrome is significantly lower than in normal subjects or patients with hemophilia B. It cannot be excluded that the frequently low levels of factor XII in patients with severe hemophilia are acquired and probably due to liver cell damage.Dedicated to Professor F. Hartmann, MD     

Effects of exercise training on plasma catecholamines and blood pressure in labile hypertensive subjects

Summary Plasma catecholamine concentrations (norepinephrine, NE; epinephrine, E) were measured along with heart rate (HR) and blood pressure (BP) at rest in supine (20 min) and standing (10 min) positions and in response to cycle ergometer exercise (5 min; 60% estimated maximal aerobic power) in 12 hypertensive patients before and after 20 weeks of aerobic training on cycle ergometer (six males, one female) or by jogging (five males). In a control group of labile hypertensive patients (five males, two females), estimated maximal aerobic power as well as HR and BP at rest in the supine and standing positions and in response to exercise were not modified from the first to the second evaluation (43±4 vs 43±5 ml·kg^–1·min^–1). In comparison estimated maximal aerobic power significantly increased in both training groups (cycle: 38±4 to 43±4; jogging: 38±3 to 46±4 ml·kg^–1·min^–1). However HR and BP were not modified following training, except for small reductions in systolic (18.9 to 18 kPa: 142 to 135 mmHg) and diastolic pressures (13.3 to 12 kPa: 100 to 90 mmHg) (p<0.05) at standing rest in the cycle group. Changes in plasma E and NE concentrations at rest and in response to exercise were small and not consistent: plasma NE was lower at standing rest following cycle training, (559±95 vs 462±108 pg·ml^–1) but a similar reduction was observed in the control group (428±45 vs 321±28 pg·ml^–1); plasma E was lower at rest following cycle training (29±7 vs 12±8 pg·ml^–1), but was higher in response to exercise (137±24 vs 419±113 pg·ml^–1). These results are in accordance with previous reports which do not clearly demonstrate that physical training in hypertensive patients lowers BP and the activity or reactivity of the sympathetic system.     

Moderation of prekallkrein–factor XII interactions in surface activation of coagulation by protein-adsorption competition

Traditional biochemistry of contact activation of blood coagulation suggesting that anionic hydrophilic surfaces are specific activators of the cascade is inconsistent with known trends in protein adsorption. To investigate contact activation reactions, a chromogenic assay was used to measure prekallkrein (PK) hydrolysis to kallikrein (Kal) by activated factor XII (FXIIa) at test hydrophilic (clean glass) and hydrophobic (silanized glass) surfaces in the presence of bovine serum albumin (BSA). Hydrolysis of PK by FXIIa is detected after contact of the zymogen FXII with a test hydrophobic surface only if putatively-adsorbed FXIIa is competitively displaced by BSA. By contrast, FXIIa activity is detected spontaneously following FXII activation by a hydrophilic surface and requires no adsorption displacement. These results (i) show that an anionic hydrophilic surface is not a necessary cofactor for FXIIa-mediated hydrolysis of PK, (ii) indicate that PK hydrolysis does not need to occur by an activation complex assembled directly on an anionic, activating surface, (iii) confirms that contact activation of FXII (autoactivation) is not specific to anionic hydrophilic surfaces, and (iv) demonstrates that protein-adsorption competition is an essential feature that must be included in any comprehensive mechanism of surface-induced blood coagulation.     

Contact activation of kallikrein and plasmin systems of rabbit blood

Contact activation of the kallikrein—kinin system by kaolin and activation of plasmin by streptokinase can take place in rabbit blood plasma. Preliminary brief treatment of plasma with chloroform removes the factor which inhibits both these activation processes.Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 4, pp. 390–392, April, 1976     

血浆高血压因子的实验研究

本研究证实了在自发性高血压大鼠血浆中存在一种循环高血压因子。当给正常血压ＳＤ大鼠静脉注射时，可以产生血压延迟性升高，在４５分钟达高峰，６０分钟恢复至原来水平。这种因子还可提高大鼠平滑肌细胞对＾４５Ｃａ＾２＋的摄取，其摄取的时间过程与血压反应一致。由此说明血浆高血压因子不是体液中已知的加压物质，而是一种有自己独特的来源、理化性质和生理功能的有形成分。可能是原发性高血压病的发病因素之一。     

Anaphylatoxin formation by contact activation of plasma. II. Implication of properdin and an unknown plasma factor in activation by zymosan

During incubation with human serum at 37°C zymosan is coated with factors which liberate anaphylatoxin (AT) from its precursor, anaphylatoxinogen. Serum free of properdin (RP), prepared by previous absorption with zymosan at 17°C, does not generate an AT-forming system when subsequently incubated with fresh zymosan, at 37°C. Only after addition of properdin to RP does the mixture fix an effective system onto zymosan. Serum pretreated with zymosan at 37°C (zymosan-R3) is unable to subsequently coat fresh zymosan with an AT-forming system even when properdin is added. It is concluded that in addition to properdin and possibly complement factors C1, C4 and C2 an unknown serum factor (UF) is essential for the generation of AT forming activity on zymosan. UF is present in RP and in cobra venom-treated serum (cobra-R3) but absent from zymosan-R3. It is subject to decay once fixed to zymosan. Decayed zymosan complexes can be regenerated by incubating them with an appropriate source of UF, e. g. RP or cobra-R3, whereas reagents containing only early complement components and/or properdin d o not reactivate them. The results indicate that AT formation in serum by zymosan and probably other polysaccharides proceeds by a pathway different from the classical complement reaction.     

Acoustics of blood plasma on solid surfaces

We have quantified surface associated coagulation of human blood plasma with a recently developed methodological system consisting of a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), a method that measures the weight of adsorbed molecules on surfaces as a function of frequency shifts of a quartz crystal. Further, it measures the damping energy (i.e. viscoelasticity) of the adsorbed layer. Four different surfaces where studied: Heparin (Hep) surface as an active inhibitor of clot formation, titanium (Ti) surfaces that are known to activate the intrinsic pathway, polystyrene (PS) surfaces and poly(urethane urea) (PUUR) surfaces. The experiments were initiated by applying citrated human plasma at the sensor surfaces; calcium was then added to initiate coagulation. The Hep surfaces showed no apparent indication of clot formation during one hour of incubation at room temperature. However, on Ti surfaces we observed an early and rapid change in both frequency shift and viscoelastic properties of the coagulating plasma. We inhibited the intrinsic pathway activation by using corn trypsin inhibitor (CTI), which is specific for factor FXIIa in the bulk phase, which prolonged the coagulation times for all non-heparinized surfaces. We have also found a peculiar initial plasma protein interaction phenomenon on Ti surfaces. The described methodology would be very efficient for basic studies of surface associated coagulation and as a screening method for new biomaterials.     

Handling-induced changes in plasma volume and osmolality: Adrenal modulation of blood parameters

Handling, physical restraint and blood sampling procedures have been shown to alter both adrenal function and water intake in rats. However, the effect of these manipulations on plasma volume and osmolality, hydrational variables known to control water intake, has not been systematically investigated. The purpose of the present investigation was to examine the effects of handling (restraint and blood sampling) on these measures. It was found that handling produced an increase in plasma volume without affecting plasma osmolality. This handling-induced hypervolemia was abolished by adrenalectomy and adrenal demedullation. The results of this investigation demonstrate that handling alters body water compartmentalization, expanding plasma volume and that this effect appears to be mediated by epinephrine. Thus, it appears that epinephrine has a role in body fluid homeostasis and should be considered as a hormone influencing water intake.     

Interrelationships between sodium clearance,plasma aldosterone,plasma renin activity,renal hemodynamics and blood pressure in renal disease

Summary This study was designed to evaluate the role of aldosterone, glomerular filtration and blood pressure on sodium excretion in renal disease. Sodium clearance (C_Na), plasma aldosterone (PA), plasma renin activity (PRA), glomerular filtration rate (GF), paraaminohippurate clearance (C_PAH) and blood pressure were measured simultaneously in 19 normal subjects, 38 patients with benign essential hypertension, 3 with renal artery stenosis, 48 with chronic glomerulonephritis, 20 with the nephrotic syndrome, 24 with tubulo-interstitial disease and 21 with a renal homograft.C_Na was significantly depressed in patients with the nephrotic syndrome. Mean PA and PRA were increased in renal artery stenosis, but within the normal range in the other groups. C_Na correlated inversely with PA in all groups but one (tubulo-interstitial disease). C_Na correlated directly with GF in the nephrotic syndrome and with the mean blood pressure (mBP) in chronic glomerulonephritis and tubulointerstitial disease. PA correlated directly with PRA and inversely with GF or C_PAH in most groups.It is concluded that PA is an important determinant of the basal natriuresis in renal disease with the exception of tubulo-interstitial nephropathies. In the nephrotic syndrome sodium retention is largely determined by the interaction of PA and GF. In chronic nephropathies, but not in benign essential hypertension, the fractional sodium excretion is partly blood pressure-dependent. Impairment of renal function is often accompanied by a rise in PA.     

Immunization induces activation of bone marrow eosinophils required for plasma cell survival

Eosinophils not only have multiple functions as effector cells of the innate immune system but also as modulators of immune responses. As producers of cytokines required for plasma cell survival, they are essential for the long-term maintenance of plasma cells in the BM. Here we show that the activation of eosinophils both in vitro and in vivo enhances the expression of the plasma cell survival factors APRIL, IL-6, IL-4, IL-10 and TNF-α. The in vivo activation of eosinophils was independent of the type of adjuvant used for primary immunization. Although eosinophils were activated by adjuvant itself, a stable activation and a constant increase in BM eosinophils were observed only in the presence of antigen. Thus, the numbers and the quality of eosinophils were dependent on priming the adaptive immune system. With secondary immunization and re-activation of antigen-dependent memory cells, the ability of eosinophils to promote plasma cell survival was further increased. These findings suggest that in T-cell-dependent immune responses eosinophils are conditioned to support the long-term survival of plasma cells in the BM, and furthermore imply that through accelerated numbers of eosinophils, stable plasma cell survival niches are established and the long-term survival of plasma cells is ensured.     

Mechanism of supression of human peripheral blood lymphocytes by platelet activation factor

Institute of Immunology, Ministry of Health of the USSR. Research Institute of Biomedical Technology, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 7, pp. 69–71, July, 1989.