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.

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.

Prolylcarboxypeptidase independently activates plasma prekallikrein (fletcher factor).

Prolylcarboxypeptidase isoform 1 (PRCP1) is capable of regulating numerous autocrines and hormones, such as angiotensin II, angiotensin III, αMSH1-13, and DesArg(9) bradykinin. It does so by cleaving a C-terminal PRO-X bond. Recent work also indicates that the human PRCP1 activates plasma prekallikrein (PK) to kallikrein on endothelial cells through an uncharacterized mechanism. This study aims to identify PRCP1 binding interaction and cleavage site on PK. Recently, a cDNA encoding a novel splice variant of the human PRCP1 was identified. This isoform differed only in the N-terminal region of the deduced amino acid sequence. Using structural and functional studies, a combination of peptide mapping and site-directed mutagenesis approaches were employed to investigate the interaction of PRCP1 with PK. Three PRCP peptides, in decreasing order of potency, from 1) the N-terminus of the secreted protein, 2) spanning the opening of the active site pocket, and 3) in the dimerization region inhibit PRCP activation of PK on endothelial cells. Investigations also tested the hypothesis that PRCP cleavage site on PK is between its C-terminal Pro 637 (P(637)) and Ala 638 (A(638)). Recombinant forms of PK with C-terminal alanine mutagenesis or a stop codon is activated equally as wild type PK by PRCP. In conclusion, PRCP1 interacts with PK at multiple sites for PK activation. PRCP1 also enhances FXIIa activation of PK, suggesting that its activation site on PK is not identical to that of FXIIa.

Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.

Light transmission aggregometry (LTA) is the most common method used to assess platelet function. However, there is no universal standard for its performance. The Platelet Physiology Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis formed a working party of experts with the aim of producing a series of consensus recommendations for standardizing LTA. Due to a lack of investigations that directly compared different methodologies to perform LTA studies, there were insufficient data to develop evidence-based guidelines. Therefore, the RAND method was used, which obtains a formal consensus among experts about the appropriateness of health care interventions, particularly when scientific evidence is absent, scarce and/or heterogeneous. Using this approach, each expert scored as "appropriate", "uncertain" or "inappropriate" a series of statements about the practice of LTA, which included pre-analytical variables, blood collection, blood processing, methodological details, choice of agonists and the evaluation and reporting of results. After presentation and public discussion at SSC meetings, the assessments were further refined to produce final consensus recommendations. Before delivering the recommendations, a formal literature review was performed using a series of defined search terms about LTA. Of the 1830 potentially relevant studies identified, only 14 publications were considered to be actually relevant for review. Based upon the additional information, 6 consensus statements were slightly modified. The final statements were presented and discussed at the SSC Meeting in Cairo (2010) and formed the basis of a consensus document, which is the subject of the present report. This article is protected by copyright. All rights reserved.

Factor XII: new life for an old protein.

Ratnoff and his coworkers recognised that factor XII (XII) stimulates cell growth and activates mitogen-activated protein kinase. We determined the receptor(s) for this function and the consequence of this signalling pathway. Investigations show that the urokinase plasminogen activator receptor serves as the XII binding site on cultured umbilical vein endothelial cells. When XII binds, it stimulates ERK1/2 and Akt S473 phosphorylation. These events are distinct because when cell mTORC2 is absent, XII phosphorylates ERK1/2 but not Akt S473. Zymogen XII is an equal stimulator of signalling as XIIa or inhibitor-treated XIIa. Peptides from uPAR domain 2 block XII binding and ERK1/2 and Akt phosphorylation. Furthermore, antibodies to the integrins ß1 and α5 block XII signalling. Likewise, inhibitors to the EGFR block XII-induced phosphorylation events. XII stimulates cell growth and proliferation. XII induces angiogenesis ex vivo in normal aortic sprouts and in vivo in matrigel plugs in normal mice, but not in aorta from uPAR knockout mice or matrigel plugs placed into uPAR-deleted mice. Skin biopsies constitutively or in a wound nine days after injury show reduced CD31 antigen expression in specimens from XII knockout mice compared to wild-type mice. These studies indicate that XII stimulates angiogenesis, a physiologic function independent of contact activation.

Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo.

BACKGROUND: Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin - RPPGF. METHODS AND RESULTS: These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at >or=0.78, 1.6, and 1.6 microm, respectively. They competitively inhibit alpha-thrombin-induced cleavage of a chromogenic substrate at 4.4-8.2 microm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks alpha-thrombin-induced calcium flux in fibroblasts with an IC(50) of 6.9 +/- 1.2 microm. FM19 achieved 100% inhibition of threshold alpha- or gamma-thrombin-induced platelet aggregation at 8.4 +/- 4.7 microm and 16 +/- 4 microm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. CONCLUSION: FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.

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.

Overexpression of prolylcarboxypeptidase enhances plasma prekallikrein activation on Chinese hamster ovary cells.

Plasma prekallikrein (PK) complexes with its receptor, high-molecular-weight kininogen (HK), on human umbilical vein endothelial cells (HUVEC). When assembled on endothelial cells, PK is activated to plasma kallikrein independent of factor XIIa by the serine protease prolylcarboxypeptidase (PRCP, Km= 9 nM). PRCP was shown to be a PK activator when isolated from HUVEC (J Biol Chem 277: 17962-17969, 2002) and produced as a recombinant protein (Blood 103: 4554-4561, 2004). To additionally confirm that human PRCP is a physiological PK activator, PRCP was overexpressed in Chinese hamster ovary (CHO) cells. CHO cells were transfected with full-length PRCP under the control of a cytomegalovirus promoter, and CHO recombinant PRCP was expressed as a fusion protein with COOH-terminal enhanced green fluorescence protein (EGFP). The presence of recombinant PRCP in transfected CHO cells was detected by real-time RT-PCR, immunoblot, and immunoprecipitation. PRCP mRNA and PK activation were two- to threefold higher in transfected than in control CHO cells. The increase in PRCP-induced PK activation in the transfected CHO cells paralleled the increase in PRCP antigen expression, as determined by anti-PRCP and anti-green fluorescence protein antibodies. PK activation of the transfected cells was blocked by small interfering RNA to PRCP. Anti-PRCP antibody and Z-Pro-Pro-aldehyde dimethyl acetate also blocked PK activation (IC50= 0.01 and 7.0 mM, respectively). Localization of PRCP in intact cells observed via confocal microscopy and flow cytometry also confirmed overexpression of PRCP on the external membrane. These investigations independently confirm that PRCP is expressed on cell membranes and that PRCP expression increases PK activation.

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.

Assembly of high molecular weight kininogen and activation of prekallikrein on cell matrix.

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires > or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.

Hemostatic/fibrinolytic protein changes in C3H/HeN mice infected with Rickettsia conorii--a model for Rocky Mountain spotted fever.

Changes in plasma hemostatic and fibrinolytic proteins were determined during courses of a murine model of fatal and non-fatal Rocky Mountain spotted fever. C3H/HeN mice were infected with Rickettsia conorii and coagulation and histopathologic studies were performed at prescribed periods of time. A significant decrease in plasma factor VIII activity and rise in plasma factor V procoagulant activity correlated with a fatal infection. Factor VII levels were unchanged; factor XI levels dropped early in the course in the lethally infected animals, but returned to normal. Factor XII, high molecular weight kininogen, and prekallikrein levels were unchanged by the sublethal infection. Prekallikrein levels fell during the lethal infection. Antithrombin concentrations were decreased significantly in all animals, but plasma plasminogen levels did not change in either group of animals. Nonocclusive thrombi were microscopically observed rarely and only in animals surviving a sublethal infection. A fall in tissue plasminogen activator activity and a rise in plasminogen activator inhibitor activity highly correlated with a lethal outcome. Lethal infection with R. conorii is associated with primary endothelial cell injury resulting in decreased tissue plasminogen activator and increased plasminogen activator inhibitor.

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.

Expression and colocalization of cytokeratin 1 and urokinase plasminogen activator receptor on endothelial cells.

The cellular localization of human cytokeratin 1 (CK1), urokinase plasminogen activator receptor (uPAR), and gC1qR, high-molecular-weight kininogen (HK)-binding proteins on endothelial cells, was determined. CK1 was found on the external membrane of nonpermeabilized endothelial cells by immunoperoxidase staining, immunofluorescence, and transmission electron microscopy using immunogold. Human umbilical vein endothelial cells (HUVECs) had 7.2 +/- 0.2 x 10(4) specific CK1 membrane sites/cell by (125)I-F(ab')(2) anti-CK1 antibody binding. Flow cytometry studies confirmed the presence of CK1, uPAR, and gC1qR on HUVECs. On laser scanning confocal microscopy and transmission electron microscopy, CK1 and uPAR, but not gC1qR, colocalized on the cell surface of HUVECs. The HUVEC surface distribution of these proteins was distinctly different from that for von Willebrand factor. In competitive inhibition experiments, anti-CK1, anti-uPAR, or anti-gC1qR blocked both biotin-HK binding and prekallikrein (PK) activation on HUVECs with an inhibitory concentration of 50% (IC(50)) of 300 to 350 nM, 50 to 60 nM, or 35 to 100 nM, respectively. Also, antibodies to uPAR and gC1qR each inhibited 86% of kallikrein-mediated, 2-chain urokinase plasminogen activation, whereas antibodies to CK1 only inhibited 24% of plasminogen activation. On HUVECs, CK1 and uPAR, but not gC1qR, colocalized to be a multiprotein receptor complex for HK binding, PK activation, and 2-chain urokinase plasminogen activation.

Assembly and activation of HK-PK complex on endothelial cells results in bradykinin liberation and NO formation.

Prekallikrein (PK) activation on human umbilical endothelial cells (HUVEC) presumably leads to bradykinin liberation. On HUVEC, PK activation requires the presence of cell-bound high-molecular-weight kininogen (HK) and Zn(2+). We examined the Zn(2+) requirement for HK binding to and the consequences of PK activation on endothelial cells. Optimal HK binding (14 pmol/10(6) HUVEC) is seen with no added Zn(2+) in HEPES-Tyrode buffer containing gelatin versus 16--32 microM added Zn(2+) in the same buffer containing bovine serum albumin. The affinity and number of HK binding sites on HUVEC are a dissociation constant of 9.6 +/- 1.8 nM and a maximal binding of 1.08 +/- 0.26 x 10(7) sites/cell (means +/- SD). PK is activated to kallikrein by an antipain-sensitive mechanism in the presence of HK and Zn(2+) on HUVEC, human microvascular endothelial cells, umbilical artery smooth muscle cells, and bovine pulmonary artery endothelial cells. Simultaneous with kallikrein formation, bradykinin (5.0 or 10.3 pmol/10(6) HUVEC in the absence or presence of lisinopril, respectively) is liberated from cell-bound HK. Liberated bradykinin stimulates the endothelial cell bradykinin B2 receptor to form nitric oxide. Assembly and activation of PK on endothelial cells modulates their physiological activities.

Thrombostatin inhibits cyclic flow variations in stenosed canine coronary arteries.

Thrombostatins are a group of compounds based upon a breakdown product of bradykinin, RPPGF. They inhibit alpha-thrombin-induced platelet activation by binding to protease activated receptor 1 and, at a lower affinity, by interacting with thrombin's active site. After a single intravenous infusion of MAP4-RPPGF (11.58 mg/kg), its t1/2alpha was 4.5 min with a clearance of 2.0 ml/min. MAP4-RPPGF administration had a sustained antiplatelet effect, preventing gamma-thrombin-induced (12.5 nM) platelet activation for 4 h. Its antiplatelet effect summated with that of aspirin and/or clopidogrel. MAP4-RPPGF was compared with aspirin and clopidogrel in the Folts model of coronary artery thrombosis. Dogs were randomized to 3 treatment groups: aspirin 1.14 mg/kg i.v., clopidogrel 0.5 mg/kg i.v., or MAP4-RPPGF 0.77 mg/kg i.v. Cyclic flow variations (CFV) were recorded in 5 untreated dogs hourly for 3 successive hours and for 1 h before (all groups >11 CFV/h), and for 2 h after drug infusion in each of the 3 treatment groups. After 1 h drug treatment, all groups of animals had <6 CFV/h; after 2 h treatment, all had <1 CFV/h. All agents significantly reduced CFV from control at each hour, but none was significantly better than any other. Thrombostatin was as effective as aspirin or clopidogrel in inhibiting coronary artery thrombosis in this canine model.

Developing peptide inhibitors to thrombin activation of platelets from bradykinin analogs.

Investigations identified peptide, platelet-selective thrombin inhibitors. Three peptides (MAP4-RPPGF, RGKWC and RGDWC) were relatively selective inhibitors of thrombin-induced platelet activation and calcium mobilization. MAP4-RPPGF at 35.5+/-0.03 microM inhibits gamma-thrombin-induced platelet aggregation 100% and alpha-thrombin-induced calcium mobilization in fibroblasts 84%. RGKWC at 800+/-400 microM inhibits gamma-thrombin-induced platelet aggregation 100% and calcium mobilization 63%. RGDWC at 140+/-100 microM inhibits gamma-thrombin-induced platelet aggregation 100% and calcium mobilization 32%. RGDWC also inhibits ADP-induced platelet aggregation, whereas MAP4-RPPGF and RGKWC do not. RGKWC prolongs the activated partial thromboplastin time (APTT) but not the prothrombin time (PT) or thrombin clotting time (TCT). RGKWC uniquely inhibits alpha-thrombin activation of human factor XI. Single amino acid substitutions in peptide pentamers result in differences in potency and mechanism(s) of inhibition of platelet and fibroblast activation by thrombin.

Plasma kallikrein/kinin system: a revised hypothesis for its activation and its physiologic contributions.

Recent studies indicate that assembly of high molecular weight kininogen on its multiprotein receptor allows for prekallikrein activation. On endothelial cells, factor XII activation is secondary to prekallikrein activation and amplifies it. The immediate consequence of plasma prekallikrein activation is the cleavage of high molecular weight kininogen (HK) with liberation of bradykinin. Cleaved high molecular weight kininogen is antiangiogenic. Bradykinin stimulates tPA liberation and nitric oxide formation. In addition, formed plasma kallikrein promotes single-chain urokinase activation and subsequent plasminogen activation. Kininogens and their breakdown products also are antithrombins. The angiotensin converting enzyme breakdown product of bradykinin prevents canine coronary thrombosis. The author presents a new hypothesis for physiologic assembly and activation of the plasma kallikrein/kinin system and discusses its influence on vascular biology.