High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets.

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.

Protease nexin-II (amyloid beta-protein precursor): a platelet alpha-granule protein.

Protease nexin-II (PN-II) [amyloid beta-protein precursor (APP)] and the amyloid beta-protein are major constituents of neuritic plaques and cerebrovascular deposits in individuals with Alzheimer's disease and Down syndrome. Both the brain and the circulation have been implicated as sources of these molecules, although they have not been detected in blood. Human platelets have now been found to contain relatively large amounts of PN-II/APP. Platelet PN-II/APP was localized in platelet alpha-granules and was secreted upon platelet activation. Because PN-II/APP is a potent protease inhibitor and possesses growth factor activity, these results implicate PN-II/APP in wound repair. In certain disease states, alterations in platelet release and processing and clearance of PN-II/APP and its derived fragments could lead to pathological accumulation of these proteins.

Assessment of whole blood coagulation with a microfluidic dielectric sensor.

Essentials ClotChip is a novel microsensor for comprehensive assessment of ex vivo hemostasis. Clinical samples show high sensitivity to detecting the entire hemostatic process. ClotChip readout exhibits distinct information on coagulation factor and platelet abnormalities. ClotChip has potential as a point-of-care platform for comprehensive hemostatic analysis. SUMMARY: Background Rapid point-of-care (POC) assessment of hemostasis is clinically important in patients with a variety of coagulation factor and platelet defects who have bleeding disorders. Objective To evaluate a novel dielectric microsensor, termed ClotChip, which is based on the electrical technique of dielectric spectroscopy for rapid, comprehensive assessment of whole blood coagulation. Methods The ClotChip is a three-dimensional, parallel-plate, capacitive sensor integrated into a single-use microfluidic channel with miniscule sample volume (< 10 µL). The ClotChip readout is defined as the temporal variation in the real part of dielectric permittivity of whole blood at 1 MHz. Results The ClotChip readout exhibits two distinct parameters, namely, the time to reach a permittivity peak (Tpeak ) and the maximum change in permittivity after the peak (Δεr,max ), which are, respectively, sensitive towards detecting non-cellular (i.e. coagulation factor) and cellular (i.e. platelet) abnormalities in the hemostatic process. We evaluated the performance of ClotChip using clinical blood samples from 15 healthy volunteers and 12 patients suffering from coagulation defects. The ClotChip Tpeak parameter exhibited superior sensitivity at distinguishing coagulation disorders as compared with conventional screening coagulation tests. Moreover, the ClotChip Δεr,max parameter detected platelet function inhibition induced by aspirin and exhibited strong positive correlation with light transmission aggregometry. Conclusions This study demonstrates that ClotChip assesses multiple aspects of the hemostatic process in whole blood on a single disposable cartridge, highlighting its potential as a POC platform for rapid, comprehensive hemostatic analysis.

Plasma kallikrein activates the epithelial sodium channel in vitro but is not essential for volume retention in nephrotic mice.

AIM: Recent work has demonstrated that activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases causes sodium retention in nephrotic syndrome. The aim of this study was to elucidate a potential role of plasma kallikrein (PKLK) as a candidate serine protease in this context. METHODS: We analysed PKLK in the urine of patients with chronic kidney disease (CKD, n = 171) and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in PKLK-deficient mice (klkb1-/- ) with experimental nephrotic syndrome induced by doxorubicin injection. RESULTS: In patients with CKD, we found that PKLK is excreted in the urine up to a concentration of 2 µg mL-1 which was correlated with albuminuria (r = .71) and overhydration as assessed by bioimpedance spectroscopy (r = .44). PKLK increased ENaC-mediated whole-cell currents, which was associated with the appearance of a 67 kDa γ-ENaC cleavage product at the cell surface consistent with proteolytic activation. Mutating a putative prostasin cleavage site in γ-ENaC prevented channel stimulation by PKLK. In a mouse model for nephrotic syndrome, active PKLK was present in nephrotic urine of klkb1+/+ but not of klkb1-/- mice. However, klkb1-/- mice were not protected from ENaC activation and sodium retention compared to nephrotic klkb1+/+ mice. CONCLUSION: Plasma kallikrein is detected in the urine of proteinuric patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site. However, PKLK is not essential for volume retention in nephrotic mice.

Platelet C1- inhibitor. A secreted alpha-granule protein.

In order to characterize which proteins of the contact phase of coagulation interact with platelets, human platelets were studied immunochemically and functionally to determine if they contain C1- inhibitor. By means of monospecific antibody to C1- inhibitor, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to measure directly platelet C1- inhibitor. With the CELISA, from 33 to 115 ng of C1- inhibitor antigen per 10(8) platelets from 15 normal donors was quantified in lysates of washed human platelets solubilized in nonionic detergent. The mean concentration in 10(8) platelets was 62 +/- 33 ng (SD). Plasma C1- inhibitor either in the platelet suspension medium or on the surface of the platelets could account for only from 6.5 to 16% of the total antigen measured in the solubilized platelets. Upon functional studies, platelets contained 84 +/- 36 ng (SD) of C1- inhibitor activity in 10(8) platelets. As assessed by the CELISA, platelet C1- inhibitor antigen was immunochemically identical to plasma and purified C1- inhibitor. In contrast, the mean concentration of platelet C1- inhibitor antigen in platelets from four patients with classical hereditary angioedema was 8.3 ng/10(8) platelets (range, 5.3 to 11.3 ng/10(8) platelets). 25 and 31% of the total platelet C1- inhibitor was secreted without cell lysis from normal platelets after exposure to collagen (20 micrograms/ml) and thrombin (1 U/ml), respectively, and this secretion was blocked by metabolic inhibitors. Platelet subcellular fractionation showed that platelet C1- inhibitor resided mostly in alpha-granules, similar to the location of platelet fibrinogen. Thus, human platelets contained C1- inhibitor, which became available by platelet secretion. The identification of platelet C1- inhibitor suggests that platelets may modulate the activation of the proteins of early blood coagulation and the classical complement pathways.

High molecular weight kininogen binds to unstimulated platelets.

Studies were performed to determine if the unstimulated platelet membrane has a site for high molecular weight kininogen (HMWK) binding. 125I-HMWK bound to unstimulated platelets. Zn++ was required for 125I-HMWK binding to unstimulated platelets and binding was maximal at 50 microM Zn++. Neither Mg++ nor Ca++ substituted for Zn++ in supporting 125I-HMWK binding to unstimulated platelets, and neither ion potentiated binding in the presence of 50 microM zinc. 125I-HMWK competed with equal affinity with HMWK for binding, and excess HMWK inhibited 125I-HMWK-platelet binding. Only HMWK, not prekallikrein, Factor XII, Factor XI, Factor V, fibrinogen, or fibronectin inhibited 125I-HMWK-platelet binding. 125I-HMWK binding to unstimulated platelets was 89% reversible within 10 min with a 50-fold molar excess of HMWK. Unstimulated platelets contained a single set of saturable, high affinity binding sites for 125I-HMWK with an apparent dissociation constant of 0.99 nM +/- 0.35 and 3,313 molecules/platelet +/- 843. These studies indicate that the unstimulated external platelet membrane has a binding site for HMWK that could serve as a surface to modulate contact phase activation.

Human neutrophils contain and bind high molecular weight kininogen.

Because plasma kallikrein activates human neutrophils, and in plasma prekallikrein (PK) circulates complexed with high molecular weight kininogen (HMWK), we determined whether HMWK could mediate kallikrein's association with neutrophils. HMWK antigen (237 +/- 61 ng HMWK/10(8) neutrophils) was present in lysates of washed human neutrophils. Little if any plasma HMWK was tightly bound and nonexchangeable with the neutrophil surface. Human neutrophils were found to possess surface membrane-binding sites for HMWK but no internalization was detected at 37 degrees C. 125I-HMWK binding to neutrophils was dependent upon Zn2+. Binding of 125I-HMWK to neutrophils was specific and 90% reversible. 125I-HMWK binding to neutrophils was saturable with an apparent Kd of 9-18 nM and 40,000-70,000 sites per cell. Upon binding to neutrophils, 125I-HMWK was proteolyzed by human neutrophil elastase (HNE) into lower relative molecular mass derivatives. Furthermore, HMWK found in neutrophils also served as a cofactor for HNE secretion because neutrophils deficient in HMWK have reduced HNE secretion when stimulated in plasma deficient in HMWK or with purified kallikrein. These studies indicate that human neutrophils contain a binding site for HMWK that could serve to localize plasma or neutrophil HMWK on their surface to possibly serve as a receptor for kallikrein and to participate in HNE secretion by this enzyme.

High-molecular weight kininogen. A secreted platelet protein.

Human platelets were studied immunochemically to determine if they contain high-molecular weight kininogen. On crossed immunoelectrophoresis with total kininogen antisera (antisera that recognizes both high- and low-molecular weight kininogen) extracts of platelets contained total kininogen antigen. Platelet total kininogen antigen showed complete antigenic identity with plasma total kininogen and displayed the same electrophoretic migration as plasma total kininogen. Using antisera monospecific to high molecular weight kininogen, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to directly measure platelet high-molecular weight kininogen. By CELISA, 27-101 ng of high molecular weight kininogen antigen per 10(8) platelets was quantitated in detergent-soluble lysates of washed human platelets from nine normal donors with a mean level of 60 ng +/- 24/10(8) platelets. Plasma high-molecular weight kininogen, either in the platelet suspending medium or on the surface of the platelets, could only account for 5% of antigen measured in the solubilized platelets. On the CELISA, platelet high-molecular weight kininogen was immunochemically identical to plasma and purified high-molecular weight kininogen. Platelet high-molecular weight kininogen was secreted from platelets after exposure to ionophore A23187 (3-15 microM), collagen (5-150 micrograms/ml), and thrombin (1.6 U/ml). Secreted platelet high-molecular weight kininogen did not become a part of the platelet Triton-insoluble cytoskeleton. On cross immunoelectrophoresis secreted platelet total kininogen antigen had a similar electrophoretic migration to plasma total kininogen. Thus, human platelets contain high-molecular weight kininogen that can be secreted from platelets and that may participate in plasma coagulation reactions.

High molecular weight kininogen is an inhibitor of platelet calpain.

Recent studies from our laboratory indicate that a high concentration of platelet-derived calcium-activated cysteine protease (calpain) can cleave high molecular weight kininogen (HMWK). On immunodiffusion and immunoblot, antiserum directed to the heavy chain of HMWK showed immunochemical identity with alpha-cysteine protease inhibitor--a major plasma inhibitor of tissue calpains. Studies were then initiated to determine whether purified or plasma HMWK was also an inhibitor of platelet calpain. Purified alpha-cysteine protease inhibitor, alpha-2-macroglobulin, as well as purified heavy chain of HMWK or HMWK itself inhibited purified platelet calpain. Kinetic analysis revealed that HMWK inhibited platelet calpain noncompetitively (Ki approximately equal to 5 nM). Incubation of platelet calpain with HMWK, alpha-2-macroglobulin, purified heavy chain of HMWK, or purified alpha-cysteine protease inhibitor under similar conditions resulted in an IC50 of 36, 500, 700, and 1,700 nM, respectively. The contribution of these proteins in plasma towards the inhibition of platelet calpain was investigated next. Normal plasma contained a protein that conferred a five to sixfold greater IC50 of purified platelet calpain than plasma deficient in either HMWK or total kininogen. Reconstitution of total kininogen deficient plasma with purified HMWK to normal levels (0.67 microM) completely corrected the subnormal inhibitory activity. However, reconstitution of HMWK deficient plasma to normal levels of low molecular weight kininogen (2.4 microM) did not fully correct the subnormal calpain inhibitory capacity of this plasma. These studies indicate that HMWK is a potent inhibitor as well as a substrate of platelet calpain and that the plasma and cellular kininogens may function as regulators of cytosolic, calcium-activated cysteine proteases.

Protease nexin-2/amyloid beta protein precursor. A tight-binding inhibitor of coagulation factor IXa.

Protease nexin-2/amyloid beta protein precursor (PN-2/A beta PP) is an abundant, secreted platelet protein which is a potent inhibitor of coagulation Factor XIa. We examined other potential anticoagulant activities of PN-2/A beta PP. Purified Kunitz protease inhibitor domain of PN-2/A beta PP and PN-2/A beta PP itself were found to prolong the coagulation time of plasma and pure Factor IXa. The Kunitz protease inhibitor domain also inhibited the ability of Factor IXa to activate Factor X. PN-2/A beta PP inhibited Factor IXa with a Ki of 7.9 to 3.9 x 10(-11) M in the absence and presence of heparin, respectively. When the second-order rate constant of PN-2/A beta PP's inhibition of Factor IXa (2.7 x 10(8) M-1min-1) was compared to that of antithrombin III (3.8 x 10(6) M-1min-1), PN-2/A beta PP was at least a 71-fold more potent inhibitor of Factor IXa than antithrombin III. PN-2/A beta PP formed a complex with Factor IXa as detected by gel filtration and ELISA. The finding that PN-2/A beta PP is a potent inhibitor of Factor IXa could help to explain the spontaneous intracerebral hemorrhages seen in patients with hereditary cerebral hemorrhage with amyloidosis Dutch-type where there is an extensive accumulation of PN-2/A beta PP in their cerebral blood vessels.

The plasma kallikrein-kinin system: its evolution from contact activation.

The plasma kallikrein-kinin system consists of the proteins factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen. It was first recognized as a surface-activated coagulation system that is activated when blood or plasma interacts with artificial surfaces. Although surface-activated contact activation occurs in vivo in the case of tissue destruction or a developing thrombus, the physiologic basis for the activation and function of this system has not been delineated. New investigations indicate that there is a proteolytic pathway on cells for PK activation independent of FXII. This pathway for PK with subsequent FXII activation indicates physiologic activities. These activities include blood pressure regulation and modulation of thrombosis risk independently of hemostasis. Furthermore, they include regulation of endothelial cell proliferation, angiogenesis and apoptosis through a cellular-based, outside-in signaling system. The present characterizations of this system, which incorrectly had been thought to initiate coagulation, represent an evolution of understanding in this field.

The contact activation and kallikrein/kinin systems: pathophysiologic and physiologic activities.

The contact activation system (CAS) and kallikrein/kinin system (KKS) are older recognized biochemical pathways that include several proteins that skirt the fringes of the blood coagulation, fibrinolytic, complement and renin-angiotensin fields. These proteins initially were proposed as part of the hemostatic pathways because their deficiencies are associated with prolonged clinical assays. However, the absence of bleeding states with deficiencies of factor XII (FXII), prekallikrein (PK) and high-molecular-weight kininogen indicates that the CAS and KKS do not contribute to hemostasis. Since the discovery of the Hageman factor 60 years ago much has been learned about the biochemistry, cell biology and animal physiology of these proteins. The CAS is a pathophysiologic surface defense mechanism against foreign proteins, organisms and artificial materials. The KKS is an inflammatory response mechanism. Targeting their activation through FXIIa or plasma kallikrein inhibition when blood interacts with the artificial surfaces of modern interventional medicine or in acute attacks of hereditary angioedema restores vascular homeostasis. FXII/FXIIa and products that arise with PK deficiency also offer novel ways to reduce arterial and venous thrombosis without an effect on hemostasis. In summary, there is revived interest in the CAS and KKS due to better understanding of their activities. The new appreciation of these systems will lead to several new therapies for a variety of medical disorders.

Quantitation and characterization of human platelet glycoprotein IIIa by radioimmunoassay.

Glycoprotein IIIa was quantitated in human platelets by radioimmunoassay using antisera specific to platelet membranes and purified glycoprotein IIIa. Glycoprotein IIIa and glycoprotein IIb were isolated from washed platelets by Triton X-114 extraction followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioiodinated glycoprotein IIIa was further purified by affinity chromatography on Lentil lectin-Sepharose 4B. Purified glycoprotein IIb showed little crossreactivity with 125I-labeled glycoprotein IIIa using the anti-platelet membrane or anti-glycoprotein IIIa antisera on a competition inhibition radioimmunoassay. The expression of glycoprotein IIIa epitopes were the same for the purified glycoprotein IIIa and glycoprotein IIIa in Triton X-100 solubilized platelets. A 66 kDa protein derived from glycoprotein IIIa by limited proteolysis of platelet membranes also expressed the same epitopes as intact glycoprotein IIIa. Solubilized platelets contained approximately 16 micrograms of total glycoprotein IIIa antigen per 10(9) cells. The level of glycoprotein IIIa determined by radioimmunoassay in one patient with Glanzmann's thrombasthenia amounted to 6.7% of normal and it was close to the values obtained by other methods.

Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid beta-protein precursor-like protein-2.

In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta-protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a approximately 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP. Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/A beta PP. These studies indicate that the KPI domains of PN-2/A beta PP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.

Modulation of the cell membrane expression of the kininogens regulates the rate of bradykinin delivery to cells.

The kininogens were first recognized as the parent molecules for bradykinin. Their relative physiologic importance in plasma hemostasis and fibrinolysis and tissue cysteine protease inhibition has not been clarifed. Recent studies on the structure and function of the plasma kininogens, their interaction with cells of the intravascular compartment, and clinical investigations on contact system activation have refocused the physiologic importance of these proteins to kinin delivery for the maintance of vasodilatory tone. Kininogen expression on platelets slows the rate of kinin liberation, and kinins upregulate kininogen expression on endothelial cells. Regulation of kinin delivery by influencing kininogen expression may provide for new agents to manipulate blood pressure.

Kininogen-cytokeratin 1 interactions in endothelial cell biology.

This review brings available data together to put in perspective the binding properties of high molecular weight kininogen (HK) to cytokeratin 1 (CK1) to direct future investigations. We summarize our knowledge of the structure and function of HK and discuss the influence of HK on the biologic activities of the plasma kallikrein/kinin system. To better understand the role of HK in regulating the activation of the kallikrein/kinin system, we discuss the interaction of HK with endothelial cell CK1 and its influence on prekallikrein activation. Last, we address the question on how a cytoskeletal protein could be a membrane-binding site and its possible role in disease states.

The contact activation system: biochemistry and interactions of these surface-mediated defense reactions.

This review is intended to be a critical state-of-the-art overview of the activation and inhibition of the proteins (factor XII, prekallikrein, high molecular weight kininogen, and factor XI) of the contact phase of coagulation. Specifically, this review will reconsider the concept of the reciprocal activation of the proteases of the contact phase of coagulation, factor XII, and prekallikrein, in light of much recent evidence indicating that factor XII, itself, autoactivates when associated with negatively charged surfaces. In addition, the mechanisms for amplification of activation of the proteins of the contact phase of coagulation will be discussed from the pivotal role of high molecular weight kininogen, or one of its altered forms, serving as a cofactor to order the activation of the zymogens it is associated with. The role and relative importance of each of the naturally occurring plasma protease inhibitors (C1-inhibitor, alpha-2-macroglobulin, alpha-1-antitrypsin, antithrombin III, and alpha-1-antiplasmin) will be assessed as they relate to the dampening of contact phase activation. Finally, the contact phase of coagulation activation will be discussed not only as a plasma proteolytic mechanism, but also as it interacts with platelets.

A multicenter clinical evaluation of the Clot Signature Analyzer.

BACKGROUND: The Clot Signature Analyzer (CSA) was designed to assess global hemostasis as a screening assay using non-anticoagulated whole blood. Three different measurements are produced by the instrument: platelet hemostasis time (PHT), clot time (CT), and collagen-induced thrombus formation (CITF). OBJECTIVES: The purpose of the present study was to determine normal ranges for these measurements and assess the performance of the CSA in patients with well-characterized hemostatic disorders and in normal subjects. PATIENTS AND METHODS: Four institutions participated in the study. Each established their own normal reference ranges. Patients with well-characterized hemostatic disorders and concurrent normal controls were subsequently examined. RESULTS: Normal ranges between institutions were similar although statistically different. One hundred and eight patients were examined: 46 individuals with von Willebrand disease (VWD) (type 1, 26; type 2A, 11; type 2B, six; type 3, three); 38 patients with a coagulation factor deficiency; 13 individuals with platelet function defects; 10 patients taking warfarin; and one individual on low-molecular-weight heparin. Of these patients, 89% had at least one abnormality by CSA: 42/46 VWD patients, 35/38 coagulation protein defect patients, 9/13 patients with platelet function defects, 9/10 patients on warfarin and 1/1 patient on low-molecular-weight heparin. Of 116 normal subjects, 103 (89%) fell within the centers' normal range. These data suggest that the CSA has a good sensitivity for bleeding disorders.

Contact activation: a revision.

In conclusion, a revised view of the contact system has been presented. This system has little to do with the initiation of hemostasis. Like lupus anticoagulants, deficiencies of contact proteins give prolonged APTTs but may be risk factors for thrombosis. BK from kininogens is a potent modulator of vascular biology inducing vasodilation, tissue plasminogen activator release, and prostacyclin liberation. Kininogens, themselves, are selective inhibitors of alpha-thrombin-induced platelet activation preventing alpha-thrombin from cleaving the cloned thrombin receptor after arginine41. Kininogens' alpha-thrombin inhibitory activity exists in intact kininogens, BK, and all of BK's breakdown products. HK also is the pivotal protein for contact protein assembly on endothelium. It is the receptor for prekallikrein which when bound to HK becomes activated to kallikrein by an endothelial cell enzyme system independent of activated forms of plasma factor XII. Prekallikrein activation on endothelial cells results in kinetically favorable single chain urokinase and plasminogen activation. Thus the "physiologic, negatively charged surface" for contact system activation is really the assembly of these proteins on cell membranes and activation by membrane-associated enzymes.

Factor XI assembly and activation on human umbilical vein endothelial cells in culture.

Biotin-FXI optimally bound to HUVEC in the presence of 40 nM high molecular weight kininogen (HK) and > or =7 microM Zn2+. There was little specific FXI binding in the absence of added HK and at concentrations of Zn2+ <15 microM. FXI and prekallikrein, but not prothrombin, blocked biotin-FXI binding to HUVEC in the presence of HK with an IC50 of 18 nM and 180 nM, respectively. Monoclonal antibody HKL16 and peptide SDD31 also inhibited biotin-XI binding in the presence of HK with an IC50 of 4.7 nM and 50 microM, respectively. Alternatively, peptide T249-F260 of FXI's apple domain 3 and heparin monosulfate were weak inhibitors of FXI binding to HUVEC. FXI bound to HUVEC with an apparent Kd of 6.9 +/- 3.0 nM and Bmax of 13 +/- 2.6 x 10(6) sites/cell. FXI bound to HK on HUVEC, but not prothrombin, became converted to FXIa. FXI activation on HUVEC resulted from tissue culture media bovine factor XIIa. HUVEC grown in human factor XI-deficient serum did not support FXI activation. FXI binding to HUVEC in culture was mostly mediated by HK and FXI activation on HUVEC is dependent on cell-associated factor XIIa.