No abstract

No abstract

A B S T R A C T An asymptomatic woman (Ms. \Vil-liams) was found to have a severe abnormality in the surface-activated intrinsic coagulation, fibrinolvtic, and kinin-generating pathways. Assays for known coagulation factors were normal while Fletcher factor (prekallikrein) was 45%, insufficient to account for the observed markedly prolonged partial thromboplastin time. Plasminogen proactivator was present at 20% of normal levels and addition of highly purified plasminogen proactivator containing 10% plasminogen activator partially corrected the coagulation and fibrinolytic abnormalities but not the kinin-generating defect. This effect was due to its plasminogen activator content. In addition, Williams trait plasma failed to convert prekallikrein to kallikrein or release kiniin upon incubation with kaolin. Kininogen

A B S T R A C T Platelets from individuals with familial hypercholesterolemia show increased sensitivity to the aggregating agents, epinephrine and ADP. Since the mechanism of this abnormal sensitivity is unknown, we examined, in vitro, the influence of the plasma lipid environment on the function of platelets. The composition of plasma lipids was altered by the addition of sonicated cholesterol-dipalmitoyl lecithin liposomes which were "cholesterol normal" (cholesterol-phospholipid mole ratio [C/P] = 1.0), "cholesterol rich" (C/P = 2.2), or "cholesterol poor" (C/P = 0). Cholesterolnormal liposomes had no influence on platelet lipids or platelet function. In contrast, after incubation for 5 h at 370C with cholesterol-rich liposomes, normal platelets acquired 39.2% excess cholesterol with no change in phospholipids or protein. The percent increase in platelet membrane cholesterol was threefold that of the granule fraction. The acquisition of cholesterol by platelets was associated with a 35-fold increase in sensitivity to epinephrine-induced aggregation (P < 0.001) and 15-fold increase to ADP aggregation (P <0.01), as determined both by aggregometry and by [14C]serotonin release. Response to thrombin or collagen was unchanged. Platelets incubated with cholesterol-poor liposomes underwent a selective loss of 21.4% cholesterol and this was associated with an 18-fold reduction in their sensitivity to epinephrine. (5,6). These studies have demonstrated that platelets from individuals with type II hyperlipoproteinemia have an increase in both platelet factor 3 availability and phospholipid content (5) and an increased sensitivity to aggregating agents, particularly epinephrine (6). However, it is not clear whether these findings are due to an intrinsic platelet defect or are related to the platelet's lipid-rich plasma environment. Furthermore, the relevance of these observations to the in vivo situation is unknown.The abnormal lipid composition of lipoproteins in certain individuals with alcoholic cirrhosis profoundly influences erythrocyte membrane lipid composition and membrane function leading to the disorder, spur cell anemia (7,8 METHODSPlatelet preparation. All blood donors were fasting for 12 h, had abstained from medications for at least 2 wk before blood donation, and had normal serum lipoproteins according to standard criteria (10), except for one who conformed to type Ha. Venous blood was collected through siliconized needles into plastic syringes and anticoagulated by mixing 9 vol of blood with 1 vol citrate-phosphate-dextrose (trisodium citrate 0.0894 M, citric acid 0.0156 M, monobasic sodium phosphate 0.0161 M, dextrose 0.1418 M). All blood processing was carried out in plastic-ware at room temperature. Platelet-rich plasma was obtained by centrifugation of samples for 10 min at 100 g. The remaining blood was centrifuged for 15 min at 1,800 g to obtain platelet-poor plasma which contained less than 20,000 platelets per A1. Platelets were counted (11) and their volume distributions measured using a Coul...

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125 I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the ␤ chain of the endothelial cell vitronectin receptor (integrin ␣ v ␤ 3 ), or fibrinogen, another ␣ v ␤ 3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties. (

The hypotension and disseminated intravascular coagulation (DIC) in bacteremia is thought to be mediated by the combined actions of cytokines, prostaglandins, and complement. The contact system, via the release of bradykinin and the activation of Factor XI, has been postulated to be contributing to the observed hypotension and DIC. Using a mAb to Factor XII (C6B7), we blocked the activation of the contact system in an established experimental baboon model in which Escherichia coli was infused to produce lethal bacteremia with hypotension. The untreated group (n = 5) displayed contact activation, manifested by a significant decrease in high molecular weight kininogen (HK) and a significant increase in a2macroglobulin-kallikrein complexes (a2M-Kal). The C6B7-treated group (n = 5) showed an inactivation of Factor XII and the changes in HK and a2M-Kal complexes were prevented. Both groups developed DIC manifested by a decrease in platelet, fibrinogen, and Factor V levels. The untreated group developed irreversible hypotension. The treated group experienced an initial hypotension that was reversed and extended the life of the animals. This study suggests that irreversible hypotension correlates with prolonged activation of the contact system, and specific antibody therapy can modulate both the pathophysiological and biochemical changes. (J. Clin. Invest. 1993.91:61-68.) Key words: septic shock * bradykinin * prekallikrein * high molecular weight kininogen * a2-macroglobulin Introduction Activation of the kallikrein-kinin system concomitant with hypotension has been demonstrated to occur in humans ( 1, 2) and in a lethal baboon model (3) during gram-negative bacteremia. The consequences ofcontact activation include the generation of kallikrein, which releases bradykinin from high molecular weight kininogen (HK),' and the generation of Factor

Prekallikrein and high-molecular-weight kininogen were found associated in normal human plasma at a molecular weight of 285,000, as assessed by gel filtration on Sephadex G-200. The molecular weight of prekallikrein in plasma that is deficient in high-molecular-weight kininogen was 115,000. This prekallikrein could be isolated at a molecular weight of 285,000 after plasma deficient in high-molecularweight kininogen was combined with plasma that is congenitally deficient in prekallikrein. Addition of purified 125I-labeled prekallikrein and high-molecular-weight kininogen to the respective deficient plasma yielded a shift in the molecular weight of prekallikrein, and complex formation could be demonstrated by incubating prekallikrein with high-molecular weight kininogen. This study demonstrates that prekallikrein and highmolecular-weight kininogen are physically associated in plasma as a noncovalently linked complex and may therefore be adsorbed together during surface activation of Hageman factor. The complex is disrupted when these proteins are isolated by ion exchange chromatography.Activation of Hageman factor upon a negatively charged surface initiates the intrinsic coagulation pathway, the fibrinolytic pathway, and the generation of the vasoactive peptide bradykinin. Recent investigations from several laboratories have shown that the proteins of the kinin-forming pathway, namely, prekallikrein (1, 2) and high-molecular-weight (HMW) kininogen (3-5), are both required for optimal activation and function of Hageman factor. Since prekallikrein and HMW kininogen are intimately associated functionally, it appeared possible that they might be physically associated in plasma. In this paper we demonstrate that prekallikrein and HMW kininogen circulate in plasma as a noncovalently linked complex. Formation of this complex was observed: (a) when prekallikrein-deficient plasma was combined with plasma deficient in HMW kininogen, (b) after prekallikrein-deficient plasma and plasma deficient in HMW kininogen were reconstituted with prekallikrein and HMW kininogen, respectively, and (c) when purified prekallikrein was incubated with HMW kininogen. Preparation of Plasma Proteins. Fresh plasma used for the isolation of prekallikrein and HMW kininogen was collected in 0.38% sodium citrate. Hexadimethrine bromide (3.6 mg) in 0.1 ml of 0.15 M saline was added for each 10 ml of blood drawn. The tubes were then centrifuged at 900 X g for 20 min at 40 and the plasma was separated with plastic pipettes. Plastic columns and test tubes were used throughout the chromatographic procedures to minimize contact activation of Hageman factor and nonspecific adsorption to glass surfaces. Samples were concentrated by ultrafiltration (Amicon Corp., Lexington, Mass.) through a UM-10 membrane. MATERIALS AND METHODSHageman Factor Fragments. Prealbumin fragments of Hageman factor were purified by chromatography of plasma on QAE-Sephadex twice, Sephadex G-100, and SP-Sephadex, and elution from alkaline disc gels after electrophoresis as rep...

Although Cl-inhibitor