High Molecular Weight Heparin Research Articles

Decrease of prothrombin level during thrombolysis in acute myocardium infarction.

Previously, the direct interactions of Bβ26-42 fibrin residues with prothrombin were demonstrated. It was also shown that forming prothrombin complexes with E- or DDE-fragments causes non-enzymatic prothrombin activation. The direct measuring of the prothrombin level in the blood plasma of patients with acute myocardial infarction (AMI) allowed us to find a situation where such an activation can occur in vivo. Blood coagulation parameters in the blood plasma of patients with AMI were measured at 2 hours, three days, and seven days after the thrombolysis by streptokinase accompanied with intravenous administration of anticoagulants: unfractionated high molecular weight heparin (HMWH) and low-molecular-weight heparin (LMWH). The prothrombin level in the blood plasma of patients with AMI was normal before thrombolytic therapy and substantially decreased after streptokinase administration. This effect was prominent in the case of concomitant anticoagulant therapy with LMWH and was not observed when HMWH was applied. It can be explained by the fact that LMWH preferentially inhibits factor Xa, while the HMWH is an effective inhibitor of both factor Xa and thrombin. This observation suggested that the prothrombin level decrease was caused by the thrombin-like activity and possible autolysis of prothrombin by thrombin. Also, thrombolytic therapy with streptokinase caused the accumulation of fibrin degradation products (FDPs), some of which were able to bind prothrombin. The dramatic decrease of prothrombin level in the blood plasma of patients with AMI during thrombolysis allowed us to conclude the non-enzymatic prothrombin activation with the following autolysis of prothrombin that contributes to the pathology.

End-on PEGylation of heparin: Effect on anticoagulant activity and complexation with protamine

Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half-life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.

Benefits and harms of direct oral anticoagulation and low molecular weight heparin for thromboprophylaxis in patients undergoing non-cardiac surgery: systematic review and network meta-analysis of randomised trials

ObjectiveTo systematically compare the effect of direct oral anticoagulants and low molecular weight heparin for thromboprophylaxis on the benefits and harms to patients undergoing non-cardiac surgery.DesignSystematic review and network meta-analysis...

Effects of Low Molecular Weight and Unfractionated Heparin on Lipoprotein Lipase and Lipid Profile in haemodialysis patients

Introduction: In haemodialysis patients, mortality from cardiovascular disease is much greater than in the general population. The current study aimed to evaluate the effects of two different types of heparin, low molecular weight heparins (LMWH) and high molecular weight heparin (HMWH), on lipid profile in patients undergoing haemodialysis.Material & Methods: A total of 60 patients on haemodialysis were selected from two main hospitals of Lahore, Pakistan and were divided into two major groups based on the type of heparin used. A 5 ml blood sample was taken from the dialysis machine to get the serum and was kept frozen at -20ºC for analysis of total cholesterol, triglycerides, high-density lipoprotein, and low-density lipoprotein. Comparison of lipoprotein lipase activity between groups were evaluated by using the student t-test. A P-value of ? 0.05 was considered statistically significant.Results: Gender wise distribution of study population (n=60) was 67% (n=40) males and 33% (n=20) females. The age distribution of individuals varied from 39-43 years. Moreover, use of HMWH was high in males (73%) as compared to female (27%) with an average age of 39 ± 12 years where duration of haemodialysis was 4.44 ± 2.83. A significant difference in LPL activity related to different times in all patients was observed. A clearer difference observed in case of LDL where LPL activity was markedly different in both groups. Our data showed that individuals using LMWH had less chances of dyslipidaemia as compared to those using HMWH.Conclusion: LMWH is a useful and safe anticoagulant during haemodialysis as compared to HMWH.

Chitosan/heparin polyelectrolyte complexes as ion-paring approach to encapsulate heparin in orally administrable SLN: In vitro evaluation

Enhancing oral bioavailability of hydrophilic drugs by encapsulation in lipid-based nanocarriers, including Solid Lipid Nanoparticles (SLN), has been well documented. In this work, high molecular weight heparin was “insolubilized” by an “ion-paring” approach, forming Chitosan/Heparin Polyelectrolyte Complexes (PEC) to promote its encapsulation in SLN. Hybrid PEC-SLN, heparin-loaded SLN (H-SLN) as well as naked PEC were prepared and characterized regarding size, Z potential, morphology, drug loading and drug release. Physicochemical characterization of the nanoparticles was also performed by differential scanning calorimetry (DSC), and Fourier Transform Infra-Red (FTIR) analysis. FITC-labeled naked PEC along with Nile Red labeled PEC-SLN were assessed on CaCo-2 cells to study cytotoxicity as well as cell internalization ability by cytometric and confocal analysis. Transepithelial electrical resistance (TEER) was measured on NCM460 cell monolayers to evaluate whether chitosan may induce a modification of tight junctions’ integrity at epithelial level. Results showed that the minimum size of PEC (around 170 nm) was at pH 5.5 with a positive surface charge and after encapsulation in SLN produced hybrid PEC-SLN with a size of about 370 nm and a negative zeta potential. In comparison to both H-SLN and naked PEC, PEC-SLN were able to achieve a pH-controlled drug release and showed on CaCo-2 cells low toxicity and rapid internalization. Finally, TEER measurements highlighted that the hybrid nanocarriers were internalized without interference in the membrane resistance. Therefore, PEC-SLN could be considered valuable candidate for further in vivo investigations about the systemic bioavailability of oral heparin.

Development of solvent-free protocols for obtaining highly substituted maleates of hyaluronic acid and other glycosaminoglycans

Solvent-free protocols using microwave-assisted heating (i) or conventional heating without additives (ii) or adding K2CO3 (iii), or triturating at room temperature in the presence of K2CO3 (iv) were first used to esterify glycosaminoglycans (GAG) with maleic anhydride. High and low molecular weight hyaluronic acid (HMW and LMW HA), dermatan sulfate (Ds), heparin (HEP) and C6-oxidized HA (carboxy-HA) were used as substrates for maleation. Protocols (i)-(iii) were most effective for obtaining maleates with high DS (1.39–2.47), but had a strong degrading effect on GAG. Protocol (iv) did not have destructive effect, but was suitable for obtaining only HMW HA maleate (DS 0.71–1.15). Primary hydroxyl groups of HA and Ds showed a higher reactivity compared to the secondary ones. A specific feature of the HEP maleation was substitution of N-sulfate groups for N-maleate groups. To demonstrate the potential of the obtained maleates for thiol-ene click-chemical strategies, the reaction with l-cysteine was performed.

Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism

Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 Å resolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of ∼10 nm involving ∼8 monosaccharides and ∼6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 m-1 s-1, its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates.

Significance, aetiology and prevention of venous thromboembolism in pregnancy and puerperium

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Engineering D-helix of antithrombin in alpha-1-proteinase inhibitor confers antiinflammatory properties on the chimeric serpin.

Antithrombin (AT) is a heparin-binding serpin in plasma which regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to being an anticoagulant, AT also exhibits antiinflammatory activities when it binds to cell surface heparan sulfate proteoglycans (HSPGs) on the endothelium via its basic residues of D-helix to elicit intracellular signalling responses. By contrast to AT, α1-proteinase inhibitor (α1-PI) is a non-heparin-binding serpin that exhibits very slow reactivity with coagulation proteases and possesses no HSPG-dependent antiinflammatory properties. To determine whether the antiinflammatory signaling specificity of AT can be transferred to α1-PI, we replaced the D-helix of human α1-PI with the corresponding sequence of human AT and expressed the chimeric serpin α1-PI/D-helix) in a bacterial expression system. High molecular weight heparin bound to α1-PI/D-helix and accelerated the inhibition of thrombin by the serpin mutant by a template mechanism reminiscent of the cofactor effect of heparin on inhibition of thrombin by AT. Like AT, α1-PI/D-helix exhibited antiinflammatory properties in both cellular and animal models. Thus, α1-PI/D-helix inhibited the barrier-disruptive effect of proinflammatory cytokines and inhibited the activation of nuclear factor-κB transcription factor in lipopolysaccharide-stimulated endothelial cells by a concentration-dependent manner. Furthermore, the chimeric serpin reduced lipopolysaccharide-mediated lethality, elicited a vascular protective effect and inhibited infiltration of activated leukocytes to the peritoneal cavity of mice in an HMGB1-mediated inflammatory model. These results suggest that grafting the D-helix of AT to α1-PI confers antiinflammatory properties on the serpin and that the chimeric serpin may have therapeutic utility for treating inflammatory disorders.

Thermodynamic characteristics of the heparin-leucine-CaCl2 system in a diluted physiological solution

Chemical equilibria in aqueous solutions of high-molecular weight heparin (Na4hep) and leucine (HLeu) are calculated through the mathematical modeling of chemical equilibria based on representative experimental pH titration data. In addition, chemical equilibria in the CaCl2-Na4hep-HLeu-H2O-NaCl system in the presence of 0.154M NaCl background electrolyte at a temperature of 37°C in the range of 2.30 ≤ pH ≤ 10.50 and initial concentrations of basic components n × 10−3 M (n ≤ 4).

Electrospinning covalently cross-linking biocompatible hydrogelators

Many hydrogel materials of interest are homogeneous on the micrometer scale. Electrospinning, the formation of sub-micrometer to micrometer diameter fibers by a jet of fluid formed under an electric field, is one process being explored to create rich microstructures. However, electrospinning a hydrogel system as it reacts requires an understanding of the gelation kinetics and corresponding rheology near the liquid–solid transition. In this study, we correlate the structure of electrospun fibers of a covalently cross-linked hydrogelator with the corresponding gelation transition and kinetics. Polyethylene oxide (PEO) is used as a carrier polymer in a chemically cross-linking poly(ethylene glycol)-high molecular weight heparin (PEG-HMWH) hydrogel. Using measurements of gelation kinetics from multiple particle tracking microrheology (MPT), we correlate the material rheology with the formation of stable fibers. An equilibrated, cross-linked hydrogel is also spun and the PEO is dissolved. In both cases, microstructural features of the electrospun fibers are retained, confirming the covalent nature of the network. The ability to spin fibers of a cross-linking hydrogel system ultimately enables the engineering of materials and microstructural length scales suitable for biological applications.

Heparin-folate-retinoic acid bioconjugates for targeted delivery of hydrophobic photosensitizers

Amphiphilic heparin-retinoic acid (HR) and heparin-folate-retinoic acid bioconjugates (HFR) were synthesized by chemical conjugation of a hydrophobic anticancer agent all-trans-retinoic acid (RA) and a targeting ligand, folic acid (FA), to the high molecular weight heparin backbone. The HR and HFR bioconjugates had a high RA content (22%, w/w) and could self-assemble into nanoparticles with efficient encapsulation of a hydrophobic photosensitizer, pheophorbide a (PhA). The HFR bioconjugate demonstrated higher PhA loading content and loading efficiency compared to HR bioconjugate. The PhA-loaded HR and HFR nanoparticles had an average diameter of about 70nm, a negatively charged surface, a sustained release pattern and self-quenching effect in a buffered solution. Furthermore, the cellular uptake of PhA-loaded HFR nanoparticles in folate receptor-positive HeLa cells was higher than that of PhA-loaded HR nanoparticles. Upon irradiation, HFR nanoparticles selectively enhanced the phototoxicity of PhA in HeLa cells while the dark-toxicity of the nanoparticles was minimal without light treatment. HFR nanoparticles also demonstrated targeted anti-cancer effect, improving the cytotoxicity of RA in HeLa cells compared to HR nanoparticles at RA concentration ≥50μg/mL. The targeting effect of HFR and PhA-loaded HFR nanoparticles was not observed in folate receptor-negative HT-29 cells. The results indicated that HFR nanoparticles may be useful for targeted delivery of hydrophobic PDT agents and as a potential nanocarrier for dual chemo-and photodynamic therapies.

Complexation in the heparin-metal ion system

The reaction of high-molecular weight heparin with Mn2+, Ni2+, Fe3+, and Co2+ in aqueous solution at 37°C with 0.154 M NaCl as the supporting electrolyte was studied by pH-metry and mathematical modeling. The formation of complex species of different compositions and stabilities was detected. The stability constants of the complexes were determined.

Arylamides: Discovery of a Novel Class of Fully-Synthetic Small Molecule Heparin and LMWH Antagonists.

AbstractAbstract 1096Structural studies of the binding interaction between the negatively charged heparin pentasaccharide binding site and antithrombin III suggested that an amphiphilic structure resembling features designed into compounds prepared for antimicrobial activity might also provide an efficient design basis for the development of heparin antagonists. A key design element of our antimicrobial approach at PolyMedix involves compounds exhibiting polycationic arrays which are appended to a diverse set of rigid backbones. A broad screening of the antimicrobial compound library in an enoxaparin (ENX) anti-factor Xa assay, coupled with subsequent secondary testing, identified compounds with potent and broad neutralizing activity. A lead compound (PMX-60056) was identified in one structural series, the salicylamides, and this compound has successfully completed Phase 1 human clinical studies. Recently, a new series of antagonists identified in the initial screen, the arylamides, was investigated for heparin neutralizing activities. Compounds were first tested for their ability to neutralize the anticoagulant activity of ENX, tinzaparin (TZP) or fondaparinux (FPX) in an in vitro amidolytic assay for factor Xa activity. Activated partial thromboplastin time (aPTT) assays demonstrated that the compounds were also active against unfractionated heparin (UFH) in a plasma-based clotting assay. The lead compound PMX150 was comparable to or better than PMX-60056 versus all anticoagulants tested (EC50s: UFH 0.6 uM, ENX 0.1 uM, TZP 0.1 uM, FPX 0.7 uM). Early compounds have shown an interesting structure activity relationship (SAR) which has identified specific regions of the molecule that are important for activity. Additionally, rotation thromboelastometry (ROTEM) shows that compounds are able to neutralize heparin and ENX in human whole blood, restoring normal coagulation profiles. As an initial test for safety, compounds were evaluated in hemolysis and cytotoxicity assays using isolated human erythrocytes, a transformed human liver cell line (HepG2 cells) and a mouse fibroblast cell line (NIH3T3). PMX150 was not cytotoxic (or hemolytic) at concentrations 180 fold greater than the EC50 concentrations in the anti-coagulation assays, indicating a high selectivity index between toxicity and efficacy. Furthermore, the SAR has identified synthetic strategies that should further improve this safety margin. The in vivo efficacy of PMX150 was evaluated in a rat coagulation model using ENX (2 mg/kg) administered by IV injection followed 3 minutes later by saline, protamine or PMX150. Blood was collected before dosing with ENX, and at 1, 3, 10, and 60 min after dosing, for aPTT and factor Xa analysis. PMX150 was more efficacious than protamine; neutralizing 89–100% of the ENX anti-factor Xa activity over the entire 60 minute time course. The in vivo assays concur with the in vitro studies, showing that compounds have been identified with high efficacy versus low molecular weight heparin (LMWH). Protamine, the heparin antagonist currently used in the clinic, routinely causes a transient decrease in blood pressure upon dosing. Hemodynamic effects with PMX-60056 have also been observed in human subjects in the absence of heparin. As PMX150 represents a new chemical series, investigations into potential adverse hemodynamic effects were performed. Blood pressure and heart rate were measured via arterial catheters in rats administered protamine or PMX150 by a 10 minute IV infusion. As expected, protamine displayed transient and prolonged blood pressure reductions at 8 and 16 mg/kg dosages, respectively. However, PMX150 had little to no effect on blood pressure at 8 mg/kg and only a minor transient reduction at 16 mg/kg. In conclusion, we have identified a new series of fully synthetic small molecule antagonists that display potent in vitro activity against UFH, the LMWHs, and fondaparinux. The lead compound in this series, PMX150 is able to neutralize ENX in vivo with more efficacy than protamine and, furthermore, has an improved hemodynamic profile with respect to protamine. Disclosures:Scott:PolyMedix, Inc.: Employment, Equity Ownership. Costanzo:PolyMedix, Inc.: Employment, Equity Ownership. Freeman:PolyMedix, Inc.: Employment, Equity Ownership. Kavash:PolyMedix, Inc.: Employment, Equity Ownership. Young:PolyMedix, Inc.: Employment, Equity Ownership. DeGrado:PolyMedix, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Jeske:PolyMedix, Inc.: Research Funding.

Sonophoresis: recent advancements and future trends

Use of ultrasound in therapeutics and drug delivery has gained importance in recent years, evident by the increase in patents filed and new commercial devices launched. The present review discusses new advancements in sonophoretic drug delivery in the last two decades, and highlights important challenges still to be met to make this technology of more use in the alleviation of diseases. Phonophoretic research often suffers from poor calibration in terms of the amount of ultrasound energy emitted, and therefore current research must focus on safety of exposure to ultrasound and miniaturization of devices in order to make this technology a commercial reality. More research is needed to identify the role of various parameters influencing sonophoresis so that the process can be optimized. Establishment of long-term safety issues, broadening the range of drugs that can be delivered through this system, and reduction in the cost of delivery are issues still to be addressed. Sonophoresis (phonophoresis) has been shown to increase skin permeability to various low and high molecular weight drugs, including insulin and heparin. However, its therapeutic value is still being evaluated. Some obstacles in transdermal sonophoresis can be overcome by combination with other physical and chemical enhancement techniques. This review describes recent advancements in equipment and devices for phonophoresis, new formulations tried in sonophoresis, synergistic effects with techniques such as chemical enhancers, iontophoresis and electroporation, as well as the growing use of ultrasound in areas such as cancer therapy, cardiovascular disorders, temporary modification of the blood-brain barrier for delivery of imaging and therapeutic agents, hormone replacement therapy, sports medicine, gene therapy and nanotechnology. This review also lists patents pertaining to the formulations and techniques used in sonophoretic drug delivery.

Gelation of Covalently Cross-Linked PEG−Heparin Hydrogels

We study PEG-heparin hydrogels to identify compositions that lead to gel formation and measure the corresponding gelation kinetics. The material consists of a maleimide-functionalized high molecular weight heparin (HMWH) backbone covalently cross-linked with bis-thiol poly(ethylene glycol) (PEG). Using multiple particle tracking microrheology, we investigate a broad composition space, defined by the number of maleimide functional sites per HMWH (f = 3.9-11.8), the molecular weight of the PEG cross-linker (M(n) = 2000, 5000, and 10 000), and the concentrations of the heparin and PEG polymers. Gelation kinetics are characterized by time-cure superposition, yielding the gel time, t(c), and the critical relaxation exponent, n. Gelation times range from 5 < t(c) ≤ 45 min, with the fastest kinetics occurring for the highest HMWH maleimide functionalities. t(c) depends nonmonotonically on the PEG cross-linker molecular weight, suggesting that gelation is affected by the length of the cross-linker relative to intermolecular interactions between heparin molecules. The critical relaxation exponent decreases from n = 0.52 for PEG 2000 to n = 0.39 for PEG 10 000. Finally, 219 equilibrated samples taken over the entire composition space are identified as liquid or solid, defining the "gelation envelope". The boundaries of this empirical gelation envelope are in good agreement with Flory-Stockmayer theory. In all, microrheological measurements enable characterization over a large parameter space and provide crucial insight into the gelation of complex, multifunctional hydrogelators used in therapeutic applications.

Collagen-based modified membranes for tissue engineering: Influence of type and molecular weight of GAGs on cell proliferation

This study aims to evaluate the effects of the two most widely used glycosaminoglycans (dermatan sulphate and heparin) on both the structural and biological properties of collagen-based modified membranes (COL/GAGs membranes) designed for tissue engineering. The molecular weight of dermatan sulphate and heparins was correlated with the membrane feasibility and the cell (fibroblasts and keratinocytes) ability to adhere and proliferate on the COL/GAG membranes. Microstructure and physico-chemical properties of COL/GAGs membranes were examined using scanning electron microscopy and differential scanning calorimetry; the free amino group content and the swelling properties were also detected. The morphology, proliferation and growth behaviour of keratinocytes and fibroblasts were investigated using microscopical approach and in vitro colorimetric assay. Both fibroblasts and keratinocytes are able to grow and proliferate on COL/dermatan sulphate membranes. Fibroblasts revealed significantly higher proliferation on the membranes prepared with heparin if compared to the proliferation on the membrane without heparin (COL membrane). Particularly, a combination of the membranes formulated adding high molecular weight dermatan sulphate and high molecular weight heparin could be suitable to be used as biomaterials for epidermal substitute.

Anticoagulant Effects of a Complex of High Molecular Weight Heparin and Arginine

Heparin forms a complex compound with arginine in a pure system, which was shown by biochemical methods. A method for obtaining the complex in vitro has been developed. At arginine/heparin molar ratio of 3:1 the complex exhibited anticoagulant, antiplatelet, and fibrin-depolymerization activities. High fibrin-depolymerization and anticoagulant activities were documented in the blood of animals 10 min after intravenous injection of the arginine-heparin complex, in contrast to the picture after injection of the complex components (heparin and arginine) alone.

Heparin affects matrix metalloproteinases and tissue inhibitors of metalloproteinases circulating in peripheral blood

Objectives Blood sampling/handling alters matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) expression. The aim of this study is to evaluate the effects of high molecular weight heparin on MMP and TIMP expression in blood. Design and methods We analyzed by gelatin zymography and ELISA assays the effects of different heparin salts, dose- and time-dependence of MMP and TIMP concentrations in plasma and sera collected with and without clot-accelerator in plastic tubes from 50 healthy donors. Results The levels and zymography of MMP-2 did not show significant changes among all samples, and during time- and dose-dependent heparin treatments. MMP-9 and TIMP-2 expression were strongly affected by heparin, with significant increase of their content and gelatinolytic activity both in time- and in dose-dependent fashion. Addition of heparin allowed also the displacement of MMP-2 prodomain, favouring zymogen activation. Conclusions Heparin has direct and indirect effects, altering MMP/TIMP complexes circulating in blood, and increasing the release of TIMP-2. To avoid misinterpretations due to MMP/TIMP complex alteration and MMP prodomain displacement, heparin should be cautiously used in blood collection procedures.

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.