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PubMed1986

Characterization of a variant prekallikrein, prekallikrein Long Beach, from a family with mixed cross-reacting material-positive and cross-reacting material-negative prekallikrein deficiency.

Studies of plasma prekallikrein in a family with prekallikrein deficiency were made. Three children had no clotting activity but approximately 35% antigen levels, and the mother and five children had twice as much prekallikrein antigen as clotting activity, suggesting the presence of a dysfunctional molecule. A nonfunctional variant form of prekallikrein was purified that contained no prekallikrein clotting activity. The variant and normal molecules were both 80,000 mol wt, immunologically indistinguishable and complexed similarly with high molecular weight kininogen. Isoelectric focusing studies suggested a difference of one charged amino acid residue. The variant was cleaved by beta-Factor XIIa 200 times slower than the normal molecule, and no amidolytic activity was detected for the cleaved variant. These data and other observations suggest that an amino acid was substituted in the variant near the NH2-terminal end of the kallikrein light chain resulting in slower cleavage by beta-Factor XIIa and the absence of enzymatic activity.

Adult↗
PubMed1996

Multiple interactive residues of recognition: elucidation of discontinuous epitopes with linear peptides.

The discontinuous epitopes of beta-factor XIIa for three mAbs were mapped by a linear peptide-based immunoblotting technique, referred to as multiple interactive residues of recognition. The Abs were incubated with a set of overlapping synthetic peptides, deduced from the cDNA sequences of beta-factor XIIa, on a polymer membrane, and the signal was amplified by an ECL assay. Several discrete sequences of the protein were recognized by each Ab. The recognized peptides were further characterized using alanine substitution analogues and peptides of different lengths. The discontinuous epitopes found for each Ab were formed by several peptides and were composed of 20 to 31 residues. The sequence FLQEA was recognized by all three Abs and was the immunodominant peptide. Abs 201/9 and 2/15 bound to very similar discontinuous sequences, but with subtle differences. The sequences 76RLHEAFSP83, 88HDLALLRLQE97, 178GFLEG182, 146FLQEA150, and 237IRE239 formed the epitope for mAb 201/9, whereas 76RLHEAFSP83, 90LALLRLQE97, 146FLQEA150, and 235AWIREHT241 formed the epitope for Ab 2/15. The third Ab 202/7 recognized the sequences 79EAFSP83, 93LRLQE97, 133WGHQF137, and 146FLQEA150. We suggest that these sequences represent the sites to which the Abs bind. This procedure provides a sensitive and convenient tool to elucidate discontinuous epitopes for the binding of Abs, receptor ligand-binding sites, or enzyme inhibitor binding sites.

Amino Acid Sequence↗
PubMed1998

In vitro blood compatibility of surface-modified polyurethanes.

Polyurethanes have proven durable materials for the manufacture of flexible trileaflet heart valves, during in vitro tests. The response of two polyurethanes of differing primary structure to parameters of blood compatibility has now been investigated, using an in vitro test cell. Platelet (beta-thromboglobulin) release, complement (C3a) activation, the activation of free plasma and surface-bound factor XII were studied using fresh, human blood (no anticoagulant) or citrated plasma in control and surface-modified polyurethane. Surface modifications were designed to affect material thrombogenicity and included covalent attachment of heparin, taurine, a platelet membrane glycoprotein fragment, polyethylene oxide (PEO), 3-aminopropyltriethoxysilane, and glucose or glucosamine. Unmodified control polyurethanes caused platelet release and complement activation. High molecular weight (2000 D) polyethylene oxide reduced platelet release slightly but only glucose attachment to the surface produced a significant reduction in platelet activation. All modifications reduced C3 activation compared with controls, but the greatest reduction was achieved with polyethylene oxide attachment or glycosylation. Most surface modifications were more activating of factor XII, both in plasma and on the material surfaces, than the control polyurethanes. Heparin and high molecular weight PEO produced the greatest activation of factor XII in the free plasma form, but low molecular weight PEO and glucosamine produced the greatest activation of surface-bound factor XIIa. The least activating surfaces, affecting both free plasma and surface-bound factor XIIa, were those treated with platelet membrane glycoprotein fragment and glucose. PEO surfaces performed relatively well, compared with controls and most surface modifications. The best overall surface, however, was the glucose-modified surface which was least activating considering all parameters of blood compatibility.

Biocompatible Materials↗
PubMed2001

Neutrophil enhancement of fibrin deposition under flow through platelet-dependent and -independent mechanisms.

We examined the effect of adherent neutrophils on fibrin deposition under laminar flow conditions. Perfusion of recalcified citrated platelet-free plasma (PFP) over neutrophils adherent to fibrinogen-coated glass at a venous wall shear rate of 62.5 s(-1) for 15 minutes resulted in dense deposition of fibrin around each neutrophil, whereas fibrin deposition on glass alone was sparse. Fibrin deposition on neutrophils was markedly reduced by anti-CD18 or anti-CD11b or a higher shear rate (250 s(-1)). Significantly less fibrin was deposited around adherent fibrinogen-coated beads, indicating that nonspecific "cross-sectional capture" effects were not responsible for the massive fibrin deposition on neutrophils. Direct visualization of fibrin capture by neutrophils and elimination of fibrin deposition at 15 minutes by a factor XIIa inhibitor (50 microg/mL corn trypsin inhibitor [CTI]) or elastase/cathepsin G inhibitors (Methoxysuccinyl-Ala-Ala-Pro-Ala-Chloromethyl-Ketone/Z-Gly-Leu-Phe-CMK, 100 micromol/L) indicated that neutrophils can capture short fibrin strands flowing in recalcified PFP lacking CTI and can also promote thrombin generation through pathways attenuated by inhibitors of factor XIIa, elastase, and cathepsin G. When neutrophils were allowed to interact with platelets on a fibrinogen surface before perfusion of recalcified CTI-treated PFP, the fibrin deposition was observed to be dramatic compared with that over surfaces coated with platelets alone or neutrophils alone and compared with that formed on platelets adherent to collagen. This neutrophil promotion of platelet-mediated fibrin formation was attenuated by inhibitors of elastase or cathepsin G but not anti-tissue factor antibody. Neutrophils can interact with platelets via released proteases to increase platelet procoagulant activity and fibrin formation in CTI-treated plasma under the low-flow conditions expected in venous thrombosis or inflammation.

Blood Coagulation↗
PubMed1986

Human plasma prekallikrein. Immunoaffinity purification and activation to alpha- and beta-kallikrein.

Prekallikrein was purified from human plasma with a final yield of 76% using as the principal step adsorption to immobilized chicken antikallikrein IgY. When purified prekallikrein (3.4 microM) was incubated in the presence of beta-Factor XIIa (0.068 microM) for 5 min at 37 degrees C and pH 7.5, alpha-kallikrein was obtained. Upon prolonged incubation (0.5-28 h), the Mr 52,000 heavy chain of alpha-kallikrein was progressively cleaved, resulting in the formation of beta-kallikrein. The formation of beta-kallikrein was characterized as an autolytic process because it was prevented by specific inhibitors of kallikrein, including aprotinin and antikallikrein antibody but not by corn trypsin inhibitor, an inhibitor specific for beta-Factor XIIa.

Animals↗
PubMed2005

Clotting time analysis of citrated blood samples is strongly affected by the tube used for blood sampling.

Our aim was to establish whether differences in clotting times for recalcified blood and plasma samples might be explained by the use of different blood collection tubes. Samples obtained from different plastic vacuum tubes were recalcified and clotting times determined by free oscillation rheometry. The clotting times for blood collected in Vacutainer (Becton Dickinson, Rutherford, New Jersey, USA) or Vacuette (Greiner Bio-One, Kremsmünster, Austria) tubes decreased with time, with maximal effect after 30 min. Blood from Monovette (Sarstedt, Nümbrecht, Germany) tubes displayed longer clotting times, which did not decrease with time. Clotting times for plasma prepared after 1 h storage in Vacutainer or Vacuette tubes were unaffected by subsequent addition of corn trypsin inhibitor to inhibit factor XIIa, although an antibody against factor XI prolonged the clotting time markedly. In Monovette plasma, both corn trypsin inhibitor and anti-factor XI effectively prolonged the clotting time. When corn trypsin inhibitor or anti-factor XI was added to the tubes before blood collection, but both additions clearly prolonged the clotting times in all types of tubes, even though corn trypsin inhibitor was less effective in whole blood. Antibodies against human tissue factor did not affect the clotting times. The amounts of platelet or leukocyte microparticles in plasma were low and similar in all tubes. This indicates that blood collection in Vacutainer or Vacuette tubes induces a rapid activation of factor XII and factor XI.

Antibodies↗
PubMed1986

The activation of pro-urokinase by plasma kallikrein and its inactivation by thrombin.

Plasma kallikrein was found to be a good activator of pro-urokinase, the inactive zymogen form of urokinase. The complete activation of pro-urokinase by plasma kallikrein was obtained in 2 h with an enzyme/substrate weight ratio of 1/30. The rate of activation of pro-urokinase by plasma kallikrein was comparable to that catalyzed by plasmin and trypsin. The rate of activation of pro-urokinase by factor XIIa was approximately one-seventh of that by plasma kallikrein. The activation of the zymogen was due to the cleavage of a single internal peptide bond, resulting in the conversion of a single chain pro-urokinase (Mr = 55,000) into two-chain urokinase (Mr = 33,000 and 22,000), and these two chains were linked by a disulfide bond(s). These results indicate an important role of plasma kallikrein for the activation of pro-urokinase in the factor XII-dependent intrinsic pathway of fibrinolysis. Thrombin also converted pro-urokinase to a two-chain form that was not activatable by plasmin, plasma kallikrein, and factor XIIa. Thrombin specifically cleaved the Arg 156-Phe 157 bond which is located 2 residues prior to the activation site of Lys 158-Ile 159.

Amino Acid Sequence↗
PubMed1988

[Changes in the kallikrein-kinin and complement system in angiography using non-ionic contrast media].

To examine alterations of the kallikrein-kinin system and of the complement due to the bolus injection of newer non-ionic contrast agents, venous blood samples were taken before and 3 min after angiography. There were no adverse contrast reactions clinically evident. Prekallikrein, kallikrein inhibition, beta-factor XIIa inhibition, C1-esterase inhibitor, C1q, C3, ATIII, HMW-kininogen, fibrinogen and factor XII were determined. Bolus injection of the contrast medium caused an activation of the kallikrein-kinin system (p less than 0.05) with reduction of prekallikrein, kallikrein-inhibition, beta-factor XIIa inhibition and C1-esterase inhibitor. The levels of C1q and C3 were also decreased (p less than 0.05) indicating an activation of the complement. Our results demonstrate, that angiography causes a significant activation of the kallikrein-kinin as well as of the complement system in spite of the use of newer non-ionic contrast agents.

Angiography↗
PubMed1988

A common neoepitope is created when the reactive center of C1-inhibitor is cleaved by plasma kallikrein, activated factor XII fragment, C1 esterase, or neutrophil elastase.

The reactive center of C1-inhibitor, a plasma protease inhibitor that belongs to the serpin superfamily, is located on a peptide loop which is highly susceptible to proteolytic cleavage. With plasma kallikrein, C1s and beta-Factor XIIa, this cleavage occurs at the reactive site residue P1 (Arg444); with neutrophil elastase, it takes place near P1, probably at residue P3 (Val442). After these cleavages, C1-inhibitor is inactivated and its conformation is modified. Moreover, in vivo, cleaved C1-inhibitor is removed from the blood stream more rapidly than the intact serpin, which suggests that proteolysis unmasks sites responsible for cellular recognition and the uptake of the cleaved inhibitor. In the study reported here, we show, using an MAb, that an identical neoepitope is created on C1-inhibitor after the cleavage of its exposed loop by plasma kallikrein, C1s, beta-Factor XIIa, and by neutrophil elastase.

Animals↗
PubMed1994

Anticoagulant and antiprotease activities of aprosulate sodium, a new synthetic polyanion, in human plasma and purified systems.

Aprosulate sodium, a new synthetic polyanion, was evaluated for anticoagulant/antiprotease activities in vitro. Aprosulate prolonged activated partial thromboplastin time (aPTT), Heptest and thrombin time (TT) with the sensitivity order of aPTT > Heptest > TT. Prothrombin time (PT) was hardly affected by the agent. Since these results indicated that inhibition of the intrinsic coagulation cascade was important for the anticoagulant activity of aprosulate, amidolytic assays were subsequently performed to characterize antiprotease activities of the agent in the common and intrinsic pathways. Aprosulate inhibited amidolytic activity of thrombin in the presence of heparin cofactor II, but was devoid of direct and antithrombin-mediated anti-factor Xa, and direct anti-factor XIIa activities. However, aprosulate was found to be a potent inhibitor of amidolytic activity of factor Xase (IXa/VIIIa). These data suggest that the direct anti-factor Xase activity of aprosulate primarily contributes to its anticoagulant activity in the intrinsic coagulation cascade.

Anticoagulants↗
PubMed2004

Heparin-level-based anticoagulation management during cardiopulmonary bypass: a pilot investigation on the effects of a half-dose aprotinin protocol on postoperative blood loss and hemostatic activation and inflammatory response.

UNLABELLED: Cardiac surgery involving cardiopulmonary bypass (CPB) leads to activation of the hemostatic/inflammatory system. We compared the influence of a half-dose aprotinin regimen on postoperative blood loss and the activation of the hemostatic/inflammatory system during CPB, when used during a heparin-level-based heparin management for cardiac surgery. Two-hundred patients (n = 100 in each group) were enrolled in this randomized prospective study. In Group I only heparin was given according to the results of the Hepcon HMS Plus. In Group II aprotinin was added with a bolus of 1 x 10(6) kallikrein inhibiting units (KIU) for the patient immediately before initiation of CPB, 1 x 10(6) KIU in the priming solution of the CPB, and a continuous infusion of 250,000 KIU/h during CPB. Postoperative blood loss was determined after 12 h. Heparin and antithrombin activity were evaluated by an anti-Xa assay and measurement of antithrombin III activity. Hemostatic activation was evaluated by adenosine diphosphate-stimulated platelet aggregometry and by measurements of the generation/release of beta-thromboglobulin (beta-TG), soluble P-selectin (sPS), thrombin (TAT), prothrombin 1 and 2 fragments (PTF1+2), factor XIIa (FXIIa), plasmin (PAP), and D-dimers. Inflammatory response was evaluated by measuring complement factors 5b-9 (C5b-9), interleukin (IL)-6, and neutrophil elastase (NE). There were no differences in the pre-CPB values or duration of CPB between the two groups. There were no differences in the post-CPB values for platelet count, platelet aggregation, beta-TG, sPS, TAT, PTF1+2, C5b-9, NE, or IL-6. The additional use of aprotinin resulted in a significant decrease of PAP, D-dimers, and 12 h postoperative blood loss, whereas generation of the contact factor XIIa was increased. The administration of aprotinin significantly reduced postoperative blood loss after cardiac surgery and CPB. This most likely has to be attributed to the antifibrinolytic effects of aprotinin. No effects on thrombin generation, platelet activation, inflammatory response, or clinical outcome were noted. IMPLICATIONS: The use of half-dose aprotinin and heparin-level-based anticoagulation management during cardiopulmonary bypass leads to a significant reduction of postoperative blood loss after cardiac surgery. This effect can most likely be attributed to the antifibrinolytic effects of aprotinin, as we did not observe effects on other variables of activation of the hemostatic/inflammatory system.

Adult↗
PubMed1986

Role of arginine residues in the coagulant activity of high molecular weight kininogen.

High molecular weight (HMW) kininogen, the cofactor for activation of the contact system of plasma proteolysis, transports and optimally positions prekallikrein and factor XI on a negatively charged surface, allowing those zymogens to be activated by surface-bound factor XIIa. HMW kininogen circulates in plasma as a procofactor that, after cleavage by kallikrein or factor XIIa, gains ability to bind to the surface. The mechanism responsible for this increased affinity for the surface is unknown. We hypothesized that modification of arginine residues may prevent cleavage of HMW kininogen, since the initial kallikrein-induced cleavage sites on the HMW kininogen molecule are at the NH2 terminal and the COOH terminal of the bradykinin-containing portion of the molecule, each of which contains arginine. We found that modification with butanedione of four arginine residues in the HMW kininogen molecule prevented bradykinin release, which results from cleavage of HMW kininogen. Furthermore, HMW kininogen coagulant activity was lost, in proportion to the degree of arginine modification, until 6.6 residues had been modified. Complex formation with prekallikrein, however, was found to be uneffected by the modification of modified HMW kininogen. To account for the loss of coagulant activity, we also examined the ability of modified HMWKa (active cofactor) to bind to an activating surface. The affinity of modified HMWKa for kaolin was tenfold less than the affinity of unmodified HMWKa. These data suggest that arginine residues play a critical role in the ability of HMW kininogen to function as an activation cofactor, both by preventing the cleavages that produce HMWKa as well as by decreasing the affinity of HMWKa for the surface.

Arginine↗
PubMed1996

The catabolism of intact, reactive centre-cleaved and proteinase-complexed C1 inhibitor in the guinea pig.

Clearance rates in the guinea pig were determined for intact guinea pig and human C1 inhibitor, the complexes of both inhibitors with human Cls, beta factor XIIa and kallikrein, and for each inhibitor cleaved at its reactive centre with trypsin. Intact human and guinea pig C1 inhibitor were cleared from the circulation more slowly (t1/2s of 9-7 h and 12.1 h and fractional catabolic rates (FCRs) of 0.09 and 0.117) than any of their cleaved or complexed forms. The reactive centre-cleaved inhibitors were cleared with half-lives of 6.75 h for humans and 10.1 h for the guinea pig. The complexes with target proteases were catabolized much more rapidly, with half-lives ranging from 3-08 h to 4.3 h. The complexes with kallikrein were cleared more slowly than those with Cls and beta factor XIIa. Complexes prepared with the guinea pig and human inhibitors were cleared at equivalent rates. The free inactivated proteases were cleared at rates similar to the equivalent complexes, except for kallikrein, which was cleared more rapidly than its complex. The fact that the complexes with different target proteases differed in their catabolism and that protease and complex catabolism were similar suggests that protease may play a direct role in clearance.

Amino Acid Sequence↗
PubMed1996

Determination of contact phase activation by the measurement of the activity of supernatant and membrane surface-adsorbed factor XII (FXII): its relevance as a useful parameter for the in vitro assessment of haemodialysis membranes.

We investigated hemodialysis membrane biocompatibility with respect to contact phase activation by determination of FXII-like activity (FXIIA) on the membrane surface and in the supernatant phase, during plasma contact with various hemodialysis membranes using an in vitro incubation test cell. The results were compared to the influence of these membranes on the activation of purified FXII. A time course for the generation of activated FXII using purified FXII solution at physiologic concentrations on two similar negatively charged polymers was performed. The membranes assessed were regenerated cellulose (Cuprophan; Akzo Faser AG, Germany), modified cellulosic (Hemophan; Akzo Faser AG), acrylonitrile-sodium methallyl copolymer-based membrane AN69S (Hospal, France), and SPAN, a new polyacrylonitrile-based copolymer (akzo Nobel AG). The plasma FXIIA at the membranes surface was significantly different between the membranes, while the supernatant phase FXIIA exhibited no significant differences. In contrast, activation of purified FXII in a plasma-free system with respect to supernatant activity indicated significant differences between the materials. A similar finding for the membrane-bound factor XIIA was also observed when purified factor XII was used. The membrane-bound FXIIA values observed in the plasma system containing heparin were significantly greater than in citrated plasma. This demonstrated the strong influence of heparin and the interaction of other plasma components to the membrane surface on the activation of contact phase of coagulation.

Adsorption↗
PubMed2004

Recombinant prolylcarboxypeptidase activates plasma prekallikrein.

The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment.

Animals↗
PubMed1999

The lack of augmentation by aspirin of inhibition of platelet reactivity by ticlopidine.

A decreased threshold for platelet activation apparently contributes to the risk of cardiovascular events, such as acute myocardial infarction. To evaluate the impact of specific agents, we characterized platelet reactivity in 9 healthy subjects before and after 5 days of ingestion of 4 commonly prescribed regimens, 81 mg of aspirin daily, 325 mg of aspirin daily, ticlopidine 250 mg twice daily, and ticlopidine plus 325 mg of aspirin daily. Platelet reactivity was assessed with (1) aggregometry induced by 4 microM adenosine diphosphate (ADP) and collagen (0.19 mg/ml) and performed in platelet-rich plasma; and (2) flow cytometric determination of ADP-induced (0.2, 0.8, and 1.5 microM) P-selectin expression in whole blood. Because anticoagulants alter platelet reactivity, results were obtained with 3 anticoagulants, citrate, enoxaparin, or corn trypsin inhibitor (CTI, a specific inhibitor of factor XIIa without effect on other coagulation factors). Ingestion of aspirin did not alter platelet activation as assessed with flow cytometry. Inhibition of the second phase of aggregation was seen with ADP-induced aggregation in platelet-rich plasma anticoagulated with citrate but not enoxaparin or CTI. Ingestion of ticlopidine led to inhibition of ADP-induced aggregation and P-selectin expression. Inhibition of platelet reactivity after the combination of aspirin and ticlopidine did not differ from ticlopidine alone. Marked interindividual variability in platelet reactivity was seen after ingestion of ticlopidine. The results indicate that assessment of effects of specific pharmacologic regimens with accurate and readily available assays of platelet reactivity may facilitate effective prophylaxis and treatment of high-risk subjects with antiplatelet regimens designed to optimally diminish platelet reactivity.

Adenosine Diphosphate↗
PubMed2001

Characterization of the H-kininogen-binding site on factor XI: a comparison of factor XI and plasma prekallikrein.

Factor XI (FXI), the zymogen of the blood coagulation protease FXIa, and the structurally homologous protein plasma prekallikrein circulate in plasma in noncovalent complexes with H-kininogen (HK). HK binds to the heavy chains of FXI and of prekallikrein. Each chain contains four apple domains (F1-F4 for FXI and P1-P4 for prekallikrein). Previous studies indicated that the HK-binding site on FXI is located in F1, whereas the major HK-binding site on prekallikrein is in P2. To determine the contribution of each FXI apple domain to HK-FXI complex formation, we examined binding of recombinant single apple domain-tissue plasminogen activator fusion proteins to HK. The order of affinity from highest to lowest is F2 F4 > F1 F3. Monoclonal antibodies against F2 are superior to F4 or F1 antibodies as inhibitors of HK binding to FXI. Antibody alphaP2, raised against prekallikrein, cross-reacts with FXI F2 and inhibits FXI-HK binding with an IC(50) of 8 nm. HK binding to a platelet-specific FXI variant lacking the N-terminal half of F2 is reduced > 5-fold compared with full-length FXI. A chimeric FXI molecule in which F2 is replaced by P2 is cleaved within P2 during activation by factor XIIa, resulting in greatly reduced HK binding capacity. In contrast, wild-type FXI is not cleaved within F2, and its binding capacity for HK is unaffected by factor XIIa. Our data show that HK binding to FXI involves multiple apple domains, with F2 being most important. The findings demonstrate a similarity in mechanism for FXI and prekallikrein binding to HK.

Alternative Splicing↗
PubMed1980

Human plasma prekallikrein. Studies of its activation by activated factor XII and of its inactivation by diisopropyl phosphofluoridate.

Human plasma prekallikrein was purified from normal plasma. The purified prekallikrein appeared homogeneous on polyacrylamide gels in the presence of sodium dodecyl sulfate and mercaptoethanol and gave two protein bands with approximate Mr 85 000. Proteolytic activation of prekallikrein by purified human beta-factor XIIa (Mr 28 000 form) resulted in the formation of kallikrein. The apparent molecular weight of kallikrein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of mercaptoethanol was identical with that of prekallikrein; reduction of kallikrein yielded a heavy chain of Mr 52 000 and two light chains of Mr 42 000 and 37 000. The appearance of kallikrein activity was directly correlated with the limited proteolysis due to beta-factor XIIa. Kinetic and immunologic studies demonstrated that plasma prekallikrein is a factor XII dependent plasminogen proactivator. The rate constant for the inactivation of prekallikrein by diisopropyl phosphofluoridate was similar to that previously reported for trypsinogen. This observation raises the possibility that low intrinsinc catalytic activity of prekallikrein may play a role in the initiation of the intrinsic blood coagulation pathway.

Amino Acids↗