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PubMed1987

Immunoblotting studies of coagulation factor XII, plasma prekallikrein, and high molecular weight kininogen.

Immunoblotting techniques for the qualitative and quantitative analysis of FXII, PK, and HMWK in whole plasma are presented. Sensitive, specific, and quantitative immunodetection of FXII and PK can be achieved by developing the blots with polyclonal antiserum followed by radiolabeled FXII or PK, respectively. This approach is based on the assumption that bivalent antibodies bind monovalently to the NC-bound antigen and have available binding sites to bind radiolabeled antigen derived from the fluid phase. This radiolabeled antigen overlay principle may be generally useful for immunodetection of any trace protein in complex mixtures, provided that the radiolabeled purified antigen is available. Immunoblotting may also be helpful for the partial characterization of the structural or functional abnormalities of CRM-positive variant molecules. For example, earlier studies of a FXII-variant molecule that had been purified and characterized were supported by immunoblotting studies of the CRM-positive deficient plasma. Quantitative measurement of HMWK is possible using a monoclonal antibody directed against the light chain of HMWK followed by radiolabeled secondary antibody. Quantitation of cleaved and single-chain HMWK is possible using dilutions of dextran sulfate-activated NHP on unreduced SDS-PAGE and dilutions of unactivated NHP with reduced SDS-PAGE as standards. These assays allow assessment of the degree of in vivo activation of the contact system in various disease states.

Antibodies, Monoclonal↗
PubMed2002

Juvenile xanthogranuloma of the oral cavity in children: a clinicopathologic study.

PURPOSE: This clinicopathologic study describes the histopathologic, immunocytochemical, and electron microscopic features of oral juvenile xanthogranuloma (JXG) in children. DESIGN: The study population consisted of 5 children (5 months to 10 years of age) with biopsy-proven JXGs obtained for consultation. RESULTS: Lesion sites were gingiva, buccal mucosa, and tongue and were described by the clinicians as (1). irritation fibroma; (2). granulation tissue; (3). pedunculated granular nodule; (4). papilloma-like lesion; and (5). brown-red umbilicated papule. Tissue was available for histopathologic (n = 5), immunocytochemical (n = 5), and ultrastructural (n = 3) studies. Three cases showed early JXG lesions possessing abundant histiocytes, but lacking Touton giant cells. The other 2 cases had classic JXG lesions with vacuolated histiocytes and Touton giant cells. Immunocytochemical findings were (1). CD68 (KP1, PGM1), moderate to diffuse; (2). fascin, moderate to diffuse; (3). factor XIIIa, focal to diffuse; (4). alpha-1-antitrypsin, moderate to diffuse; (5). S-100 protein, focal to diffuse; and (6). CD1a, negative in all cases. Ultrastructural examination displayed histiocytic and dendritic cells lacking pentalaminar structures (Birbeck granules). CONCLUSION: JXGs of the oral cavity vary in their clinical and histopathologic appearances and necessitate that Langerhans' cell disease (LCD) be excluded. JXG and Langerhans' cell disease may occur concurrently, before or after each other, in some children.

Antigens, CD↗
PubMed2002

Identification and characterization of prolylcarboxypeptidase as an endothelial cell prekallikrein activator.

Our recent investigations have postulated a human umbilical vein endothelial cell (HUVEC)-associated prekallikrein activator (PKA). When prekallikrein (PK) assembles on high molecular weight kininogen on HUVEC, PK is activated to kallikrein. PKA was found in the 15,800 x g pellet of HUVEC lysates using an assay that measures PK activation only when bound to high molecular weight kininogen linked to microtiter plates. Sequential DEAE, wheat germ lectin affinity, and hydroxyapatite chromatography resulted in four protein bands on SDS-PAGE. One protein in the 73-kDa band was identified by amino acid sequencing as prolylcarboxypeptidase (PRCP). On gel filtration, PKA activity was a single homogenous peak identical in migration to the 73-kDa immunoblot of PRCP. Anti-PRCP inhibits PKA activity and PK activation on HUVEC. Purified PKA was blocked by diisopropyl fluorophosphate (1 mm), phenylmethylsulfonyl fluoride (3 mm), leupeptin (100 microm), antipain (IC(50) = 2 microm), HgCl(2) (IC(50) = 500 microm), Z-Pro-Pro-aldehyde-dimethyl acetate (IC(50) = 1 microm), and corn trypsin inhibitor (IC(50) = 40 nm). PKA did not correct the coagulant defect in factor XII deficient plasma, was purified from HUVEC cultured in factor XII-deficient serum, was not detected by antibody to factor XII, did not activate FXI, and was not inhibited by a neutralizing antibody to FXII. Angiotensin II (IC(50) = 2 microm) or bradykinin (IC(50) = 100 microm), but not angiotensin II-(1-7) or bradykinin(1-5), and the prolyl oligopeptidase inhibitor Fmoc-Ala-Pyr-CN (IC(50) = 50 nm) also blocked purified PKA activation of PK. The K(m) of PK activation by PRCP is 6.7 nm. PRCP antigen is present on the membrane of fixed but not permeabilized HUVEC. PRCP appears to be a HUVEC-associated PK activator.

Angiotensin II↗
PubMed2005

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays.

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.

Amino Acid Sequence↗
PubMed2000

The link between high-fat meals and postprandial activation of blood coagulation factor VII possibly involves kallikrein.

Contrary to low-fat meals, high-fat meals are known to cause postprandial factor VII (FVII) activation, but the mechanism is unknown. To study the postprandial FVII activation in detail, 18 young men consumed in randomized order high-fat or low-fat test meals. Fasting and non-fasting blood samples were collected. The high-fat test was associated with an increase in plasma triglyceride and kallikrein concentrations and postprandial FVII activation (p<0.001). Plasma kallikrein was strongly associated with triglycerides in fasting and non-fasting samples (r2=0.74-0.87, p<0.0001), suggesting that triglyceride-rich lipoproteins may activate prokallikrein. Neither plasma triglycerides nor kallikrein and activated FVII were statistically associated. This may suggest that additional factors are involved in the postprandial FVII activation. No clear evidence for a role of tissue factor expression by monocytes, factor XII or insulin in postprandial FVII activation was observed. Tissue factor pathway inhibitor and prothrombin fragment 1+2, a marker of thrombin generation, were not affected postprandially after either the high-fat or the low-fat meals. Our findings indicate that triglyceride-rich lipoproteins activate prokallikrein postprandially, which might form an important initial event in FVII activation after consumption of high-fat meals.

Adult↗
PubMed2005

Defective thrombus formation in mice lacking coagulation factor XII.

Blood coagulation is thought to be initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. In contrast, coagulation factor XII (FXII)-mediated fibrin formation is not believed to play an important role for coagulation in vivo. We used FXII-deficient mice to study the contributions of FXII to thrombus formation in vivo. Intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in the formation and stabilization of platelet-rich occlusive thrombi. Although FXII-deficient mice do not experience spontaneous or excessive injury-related bleeding, they are protected against collagen- and epinephrine-induced thromboembolism. Infusion of human FXII into FXII-null mice restored injury-induced thrombus formation. These unexpected findings change the long-standing concept that the FXII-induced intrinsic coagulation pathway is not important for clotting in vivo. The results establish FXII as essential for thrombus formation, and identify FXII as a novel target for antithrombotic therapy.

Animals↗
PubMed1998

Postprandial effects of an oleic acid-rich oil compared with butter on clotting factor VII and fibrinolysis in healthy men.

BACKGROUND: Factor VII coagulant activity (FVII:c) is associated with an increased risk of fatal ischemic heart disease, is correlated with plasma triacylglycerol concentration, and increases after a meal rich in long-chain fatty acids. OBJECTIVE: We planned to compare effects of meals rich in oleate and butter fat with those of a low-fat meal on FVII:c and fibrinolytic activity. DESIGN: A crossover design was used to compare the postprandial effects on coagulant and fibrinolytic activities in 12 men of 3 high-fat (95 g) meals--high oleate, butter, and oleate + medium-chain triacylglycerols (oleate+MCT)--with an isoenergetic low-fat meal (18 g MCT). The oleate+MCT blend was used to mimic the ratio of long-chain to shorter-chain fatty acids in butter. RESULTS: Neither the amount nor type of fat consumed influenced plasminogen activator inhibitor 1 or t-plasminogen activator activities or D-dimer concentration. FVII:c increased by 12.5% (95% CI: 4.6%, 20.5%) after the high-fat meals at 3 h and by 6.7% (95% CI: 1.6%, 11.7%) at 7 h and changed 7 h after the low-fat meal by -14.3% (95% CI: -3.3%, -25.4%). The responses to the high-fat meals did not differ. Measurements of activated FVII (FVIIa), FVII zymogen, and activated FXII (FXIIa) concentrations made after the low-fat and high-oleate meals showed a significant increase in FVIIa only after the high-oleate meal. CONCLUSIONS: The results of this study confirm that FVII:c falls after a low-fat meal and suggests that postprandial activation of FVII occurs rapidly after a fat-rich meal without involving an increase in FXIIa.

Adolescent↗
PubMed1984

Inhibition of the cold activation of Factor VII and the prothrombin time.

Normal whole blood, collected and stored in borosilicate or commercial siliconized borosilicate tubes at 4 degrees C, undergoes a time-dependent activation of Factor VII and a shortening of the prothrombin time (PT). Addition of inhibitors to Factor XII fragments (corn Hageman factor inhibitor, CHFI) or to activated Factor XII (Cytochrome-C) inhibited in a concentration-dependent way the cold-promoted activation of Factor VII and the shortening of the PT. When celite was added to whole blood in polypropylene tubes at 4 degrees C, Factor VII was activated and the PT shortened. Preincubation of celite with CHFI or Cytochrome-C prevented the activation of Factor XII and the subsequent activation of Factor VII and the PT. These studies demonstrate the importance of Factor XII in the cold activation of Factor VII and shortening of the PT and indicate that inhibitors to activated Factor XII or XII fragments are useful in inhibiting in vitro shortening of the PT. These findings suggest that the development of a collection tube that prevents contact activation also would inhibit adequately the cold-promoted activation of the PT and Factor VII.

Aprotinin↗
PubMedDate not supplied

Comparative studies of impurities in intravenous immunoglobulin preparations.

In a comparative study, seven commercially available immunoglobulin preparations and one experimental preparation, processed by various manufacturers in the United States and Europe, were investigated for non-IgG proteins and impurities. The basic fractionation methodology affects the purity of subsequently processed intravenous preparations. Five of the seven products manufactured in Europe contained varying amounts of plasma proteins other than IgG. A residual heterologous protein was detected and quantitated in one product whose processing includes porcine pepsin treatment. Only one of the eight products was low in aggregates (as judged by both anticomplement activity and nephelometric turbidity), while three products contained anticomplement activity at levels high enough to be a potential cause of reactions in patients. In general, the level of prekallikrein activator activity was proportional to that of amidolytic activity; four products had only trace levels of amidolytic activity as well as prekallikrein activator. Only one product contained significant isoagglutinin levels, while six products had detectable IgA levels.

Complement Inactivator Proteins↗
PubMed2005

Contribution of the N-terminal and C-terminal domains of haemaphysalin to inhibition of activation of plasma kallikrein-kinin system.

Haemaphysalin is a kallikrein-kinin system inhibitor from hard tick Haemaphysalis longicornis, and consists of two Kunitz type protease inhibitor domains. Each domain as well as haemaphysalin inhibited intrinsic coagulation by inhibiting activation of the kallikrein-kinin system without affecting the amidolytic activities of intrinsic coagulation factors, indicating that both domains were involved in the inhibition through a similar mechanism to that for haemaphysalin. Reconstitution experiments showed that the C-terminal domain contributed more predominantly to this inhibition. Direct binding assaying showed that the C-terminal domain could bind to the cell-binding region of high molecular weight kininogen (HK), suggesting that it also binds to the cell-binding region of factor XII. Judging from these findings, the C-terminal domain may more effectively inhibit the association of factor XII and HK with the cell surface by binding to cell-binding regions, and hence would predominantly contribute to the inhibition of activation of the kallikrein-kinin system.

Animals↗
PubMed1991

Effect of intraperitoneal administration of two different batches of albumin solutions on peritoneal solute transport in CAPD patients.

The effects of intraperitoneal administration of two different batches of human albumin (batch A and batch B) on peritoneal solute transport and dialysate white cell count were studied in 16 CAPD patients. The studies were done on two separate days during a 4-h dwell, one day without and one day with the intraperitoneal administration of 10 g/l human albumin. Marked differences were found between the two batches. The transport of all measured solutes increased during administration of batch A compared to the control experiments: urea 78% +/- 62%, lactate 51% +/- 38%, creatinine 96% +/- 54%, glucose 67% +/- 55%, inulin 27% +/- 33% IgG 126% +/- 80%, mean +/- SD; P less than 0.02). In the experiments with batch A the white cell count of the test bag was greater than that of the effluent ('night bag') before the test (13 +/- 5 vs 194 +/- 61 mm3/l; P less than 0.02). These effects were also observed when the dialysate was buffered to pH = 7.4 before inflow. Albumin batch B showed no effect on solute transport and white cell count. The effects of solute transport and white cell count are probably caused by the greater concentration of prekallikrein activator in batch A when compared to batch B (30.1 vs 0 U/l). Caution is warranted when human albumin is used for simultaneous measurement of peritoneal fluid and solute kinetics.

Absorption↗
PubMed1994

Factor-XIIIa-positive cells in normal peripheral nerves and cutaneous neurofibromas of type-1 neurofibromatosis.

Cutaneous neurofibromas consist of heterogenous cell populations including Schwann cells, perineurial cells, and fibroblastlike cells. However, the histogenesis of neurofibromas, particularly the origin and nature of the fibroblastlike cells, is still controversial. Recently, cells containing blood coagulation factor XIIIa have been reported in cutaneous neurofibromas, although their identity is uncertain. In this report, by the combination of double immunohistochemical staining and immunoelectron microscopy, we demonstrate that factor-XIIIa-positive cells are distinct from Schwann cells, perineurial cells, endothelial cells, mast cells, and conventional macrophages, but correspond to the fibroblastlike cells in cutaneous neurofibromas. Such factor-XIIIa-positive cells in cutaneous neurofibromas, however, differ from conventional fibroblasts in the strong expression of HLA-DR antigen and lack of prolyl 4-hydroxylase. Similarly, so-called endoneurial fibroblasts and, occasionally, connective tissue cells within perineurium and epineurium in normal peripheral nerve fibers express factor XIIIa as well as HLA-DR antigen. The results suggest that fibroblastlike cells in cutaneous neurofibromas are probably derived from factor-XIIIa- and HLA-DR antigen-positive connective tissue cells in peripheral nerves. The role of such factor-XIIIa-positive cells in the growth and development of cutaneous neurofibromas is discussed.

Actins↗
PubMed1992

Aprotinin in cardiopulmonary bypass--effects on the Hageman factor (FXII)--Kallikrein system and blood loss.

Aprotinin has been used in our hospital in open heart surgery for almost 20 years and recently published studies have revealed a reduction in postoperative blood loss under this therapy. In the present study patients undergoing aorto-coronary bypass operations received either 20,000 KIU aprotinin/kg body weight (group 2) or 60,000 KIU aprotinin/kg body weight (group 3). Another group of patients without aprotinin served as a control (group 1) and postoperative bleeding was more pronounced in these patients compared with the other groups. In parallel, slight reductions in kallikrein-like activity were observed in patients treated with aprotinin. Furthermore, we have shown that the main inhibitor of the contact phase, C1-esterase-inhibitor, loses some of its activity against beta-FXIIa in the presence of heparin. Aprotinin was able to partly antagonize this phenomenon. All studies dealing with the effect of aprotinin in extracorporeal circulation demonstrate hyperfibrinolysis in untreated patients. Aprotinin is known to inhibit plasmin at low concentrations and thus reduced the postoperative bleeding tendency (group 2). In addition, plasma kallikrein is inhibited by high aprotinin concentrations and is responsible for a reduced activation of the contact phase system. This effect led to a further reduction in blood loss (group 3).

Aprotinin↗
PubMed1996

Measurement of activated factor XII in health and disease.

We investigated a new ELISA for measuring activated factor XII (FXIIa) in plasma. The intra-assay coefficient of variation was 3.5% and 5.1% for plasma containing 2.5 and 8.2 ng/ml FXIIa. The inter-assay coefficient of variation was 6.2% and 6.6%. FXIIa correlated with age in women older than 55 years (r = 0.55, P = 0.0003). Mean levels in the whole population of 160 healthy individuals included in this study were not different between men and women, but women younger than 55 years had lower levels than older women and men of the corresponding age. In a group of 25 healthy centenarians FXIIa was significantly higher (3.2 ng/ ml, 95% CI 2.3-3.6) than in controls (2.1 ng/ml, 95% CI 1.8-2.4). Increased levels were also found in pregnancy, with higher levels in the third trimester (4.7 ng/ml, 95% CI 3.9-5.5) than in the first trimester (2.9 ng/ml, 95% CI 2.2-3.9). FXIIa was unmeasurable in patients with FXII deficiency, but normal in patients with FXI deficiency and C1-inhibitor deficiency. FXIIa was significantly higher than in normal controls in patients with severe sepsis (3.9 ng/ml, 95% CI 2.8-5.4) and septic shock (5.4 ng/ml, 95% CI 3.7-7.7). After treatment with thrombolytic agents, a marked increase of FXIIa was found in patients with myocardial infarction. In conclusion, the immunoassay of FXIIa permits to study more directly the contact phase of blood coagulation in situations in which the involvement of this system may play a pathophysiological role.

Adult↗