Endogenous Heparin Research Articles

A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2

Plasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) after sensing viral/bacterial RNA or DNA by toll-like receptor (TLR) 7 or TLR9, respectively. However, aberrant pDCs activation can cause adverse effects on the host and contributes to the pathogenesis of type I IFN-related autoimmune diseases. Here, we show that heparin interacts with the human pDCs-specific blood dendritic cell antigen 2 (BDCA-2) but not with related lectins such as DCIR or dectin-2. Importantly, BDCA-2-heparin interaction depends on heparin sulfation and receptor glycosylation and results in inhibition of TLR9-driven type I IFN production in primary human pDCs and the pDC-like cell line CAL-1. This inhibition is mediated by unfractionated and low-molecular-weight heparin, as well as endogenous heparin from plasma, suggesting that the local blood environment controls the production of IFN-α in pDCs. Additionally, we identified an activation-dependent soluble form of BDCA-2 (solBDCA-2) in human plasma that functions as heparin antagonist and thereby increases TLR9-driven IFN-α production in pDCs. Of importance, solBDCA-2 levels in the serum were increased in patients with scrub typhus (an acute infectious disease caused by Orientia tsutsugamushi) compared to healthy control subjects and correlated with anti-dsDNA antibodies titers. In contrast, solBDCA-2 levels in plasma from patients with bullous pemphigoid or psoriasis were reduced. In summary, this work identifies a regulatory network consisting of heparin, membrane-bound and solBDCA-2 modulating TLR9-driven IFN-α production in pDCs. This insight into pDCs function and regulation may have implications for the treatment of pDCs-related autoimmune diseases.

Influence of Peptide Bioregulators on Indicators of Hemostasis in Blood of Irradiated Experimental Animals at Low Altitude Conditions

In modern biology and medicine, much attention is paid to the study of the hemostasis regulation of some cell populations by substances of a peptide nature. They have the ability to regulate the functional and proliferative activity of cells and ensure communication in normal and pathological conditions. Research data showed that significant violations of the hemostatic system also occur with radiation damage to the body. One of the first radiation responses involves the coagulation cascade's activation, which leads to the breakdown of fibrinogen and the formation of fibrin clots. The potential threat of this is quite evident due to the existence of uranium mining tailings in several regions of the Kyrgyz Republic. Considering that significant disturbances of the hemostatic system occur during radiation damage to the body, and effective means are clearly not enough, the study of the effect of bioregulatory peptides in these conditions is of great importance. In the study, we carried out the total X-ray irradiation of laboratory animals on the X-ray therapeutic apparatus RUM-17. Peptide bioregulators peptide-1 and peptide-2 were administered intramuscularly to irradiated animals. Throughout the research, we used methods characterizing all links of hemostasis: vascular-platelet hemostasis, coagulation hemostasis, and fibrinolysis. The study has shown that the use of the peptide bioregulators in the background of acute radiation pathology leads to a decrease in the intensity of the chronic course of post-radiation thrombo hemorrhagic syndrome, contributing to an increase in the content of antithrombin III in the blood and modulating the versatile effects of endogenous heparin, which undoubtedly have a beneficial effect on the pathogenesis of radiation sickness.

Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

The cell surface serine protease Transmembrane Protease 2 (TMPRSS2) is required to cleave the spike protein of SARS-CoV-2 for viral entry into cells. We determined whether negatively-charged heparin enhanced TMPRSS2 inhibition by alpha-1-antitrypsin (AAT). TMPRSS2 activity was determined in HEK293T cells overexpressing TMPRSS2. We quantified infection of primary human airway epithelial cells (hAEc) with human coronavirus 229E (HCoV-229E) by immunostaining for the nucleocapsid protein and by the plaque assay. Detailed molecular modeling was undertaken with the heparin–TMPRSS2–AAT ternary complex. Enoxaparin enhanced AAT inhibition of both TMPRSS2 activity and infection of hAEc with HCoV-229E. Underlying these findings, detailed molecular modeling revealed that: (i) the reactive center loop of AAT adopts an inhibitory-competent conformation compared with the crystal structure of TMPRSS2 bound to an exogenous (nafamostat) or endogenous (HAI-2) TMPRSS2 inhibitor and (ii) negatively-charged heparin bridges adjacent electropositive patches at the TMPRSS2–AAT interface, neutralizing otherwise repulsive forces. In conclusion, enoxaparin enhances AAT inhibition of both TMPRSS2 and coronavirus infection. Such host-directed therapy is less likely to be affected by SARS-CoV-2 mutations. Furthermore, given the known anti-inflammatory activities of both AAT and heparin, this form of treatment may target both the virus and the excessive inflammatory consequences of severe COVID-19.

TMEM184A: Evidence for a Potential Link Between HSPGs and Mechanotransduction

Heparin is a known anticoagulant that, with cellular uptake, contributes to a variety of physiological conditions, including experimentally observed decreases in vascular cell proliferation and inflammation. The fact that heparin is endocytosed and decreases cellular responses rather than blocking them entirely suggested a receptor mediated model, which has been previously identified in our laboratory as TMEM184A. Our data suggest that TMEM184A associates with vascular endothelial‐cadherin (VE‐Cadherin) and heparan sulfate proteoglycans (HSPGS), modulating mechanotransduction events in response to heparin treatment and fluid shear stress (FSS), which replicates the stress induced by blood flow under physiological conditions. TMEM184A seems to play a role in trafficking HSPGs such as syndecan‐1 (SDC1) and glypican‐1 (GPC1), both of which have been characterized as mechanosensing proteoglycans. In recent studies it has been demonstrated that VE‐Cadherin may interact with HS chains. Data in the literature have shown that when HS chains are degraded by Heparinase III treatment, the strain induced decrease in VE‐Cadherin is abolished, indicating that the HSPGS are crucial in sensing the strain. Given these findings, as well as our preliminary evidence that TMEM184A associates with HSPGs, we hypothesize that TMEM184A acts as an interface in the mediation of mechanosignaling events on the extracellular surface of adherens junctions. Co‐immunoprecipitation pull down of TMEM184A followed by subsequent western blotting and immunostaining showed both VE‐Cadherin and TMEM184A in the bound supernatant, indicating that the two may colocalize at endothelial adherens junctions and associate in a mechanosensing complex. Using a GFP‐TMEM184A plasmid construct to overexpress TMEM184A in bovine aortic endothelial cells (BAOECS), our research team observed increased surface levels of VE‐Cadherin in adherens junctions under static flow conditions. These overexpressing cells were subjected to FSS, which increases TMEM184A associations with HSPGs. Our team observed an additive increase in VE‐Cadherin in adherens junctions when compared to overexpression alone in static conditions. An additional experiment done to elucidate the role of endogenous heparin (HSPGs) revealed that BAOECs washed with a concentrated salt buffer to remove HPSG‐protein interactions demonstrated decreased colocalization of VE‐Cadherin, TMEM184A, and various vesicle associated membrane proteins. The addition of heparin rescued these colocalization events, implicating HSPGs in this complex. Our ongoing work aims to elucidate how associations of TMEM184A with VE‐cadherin and HSPGs facilitate activation of mechanically induced signals and mediate the trafficking of adherens junctions’ surface proteins.Support or Funding InformationHL54269 to LJLK

Potential Mechanisms of Cancer-Related Hypercoagulability.

The association between cancer and thrombosis has been known for over a century and a half. However, the mechanisms that underlie this correlation are not fully characterized. Hypercoagulability in cancer patients can be classified into two main categories: Type I and Type II. Type I occurs when the balance of endogenous heparin production and degradation is disturbed, with increased degradation of endogenous heparin by tumor-secreted heparanase. Type II hypercoagulability includes all the other etiologies, with factors related to the patient, the tumor, and/or the treatment. Patients with poor performance status are at higher risk of venous thromboembolism (VTE). Tumors can result in VTE through direct pressure on blood vessels, resulting in stasis. Several medications for cancer are correlated with a high risk of thrombosis. These include hormonal therapy (e.g., tamoxifen), chemotherapy (e.g., cisplatin, thalidomide and asparaginase), molecular targeted therapy (e.g., lenvatinib, osimertinib), and anti-angiogenesis monoclonal antibodies (e.g., bevacizumab and ramucirumab).

Влияние гепарина и этанола на активность лактатдегидрогеназы в митохондриальной и цитоплазматической фракциях клеток печени крыс

Experiments on rats have shown that ethanol at a dose of 4.5 g/kg leads to an increase in the activity of lactate dehydrogenase (LDH) in rat liver cells in a direct reaction in subcellular fractions. In the cytoplasmic fraction, the LDH activity in the reverse reaction decreased. Pre-administration of heparin to animals at a dose of 250 IU/kg partially neutralizes the effects of intraperitoneal administration of ethanol, which is expressed in a decrease in the activity of LDH in rat liver cells in a direct reaction in subcellular fractions. Binding of endogenous heparin to protamine sulfate and subsequent administration of ethanol leads to an increase in LDH activity in the mitochondrial fraction of rat liver cells.

Features of local pulmonary hemostasis in patients who received surgical treatment for benign peripheral lung tumors

Objectives - to develop a method for prevention of thromboembolic events in small branches of pulmonary artery in post-operative period, based on features of local pulmonary hemostasis in patients who underwent lung surgery for benign tumors. Material and methods. 30 male patients aged 62 ± 2.4 years old were included in the study. All patients underwent lung surgery (mainly lobectomy). Blood was drawn intra-operationally from a. pulmonale (arriving venous blood), v. pulmonale (outflowing blood) and cubital vein. The following parameters of coagulative and thrombocytic-vascular hemostasis were examined: free endogenous heparin was evaluated by protamine titration, platelet count and platelet aggregative properties were registered, using hematology analyzer, activated partial thromboplastin time (aPTT), quantitative analysis of fibrinogen and protein C was performed using CoaLAB 1000 analyzer. Results. Study of hemostasis features in patients, who underwent lung surgery, has revealed an imbalance of coagulation and anticoagulation systems, which manifested itself as dysfunctions in thrombocytic-vascular hemostasis (increase in platelets aggregative properties, most notable in peripheral venous blood), plasma hemostasis (decrease in aPTT, most notable in arterial blood, and increased fibrinogen level) and anticoagulation (modestly decreased protein C and heparin levels in peripheral blood), which is characteristic of hypercoagulation, being the possible basis for development of thromboembolic complications in post-operative period. These changes are most typical for blood taken from cubital vein, which is indicative of increased possibility of clot formation specifically in systemic circulation, which is indicative of increased possibility of clot formation specifically in systemic circulation. The least risk of clot formation was established in pulmonary vein blood (outflowing blood). Moderate correlation between free heparin level in arterial blood and venous blood was established. Conclusion. The derived results have allowed us to develop an individual management plan for patients with increased risk of thromboembolic events, which includes early administration of anticoagulant agents in prophylactic doses.

Biochemical and functional characterization of glycosaminoglycans released from degranulating rat peritoneal mast cells: Insights into the physiological role of endogenous heparin

The properties of commercially prepared heparin as an anticoagulant and antithrombotic agent in medicine are better understood than is the physiological role of heparin in its native form, where it is uniquely found in the secretory granules of mast cells. In the present study we have isolated and characterised the glycosaminoglycans (GAGs) released from degranulating rat peritoneal mast cells. Analysis of the GAGs by NMR spectroscopy showed the presence of both heparin and the galactosaminoglycan dermatan sulphate; heparinase digestion profiles and measurements of anticoagulant activity were consistent with this finding. The rat peritoneal mast cell GAGs significantly inhibited accumulation of leukocytes in the rat peritoneal cavity in response to IL-1β (p < 0.05, n = 6/group), and inhibited adhesion and diapedesis of leukocytes in the inflamed rat cremasteric microcirculation in response to LPS (p < 0.001, n = 4/group). FTIR spectra of human umbilical vein endothelial cells (HUVECs) were altered by treatment of the cells with heparin degrading enzymes, and restored by the addition of exogenous heparin. In conclusion, we have shown that rat peritoneal mast cells contain a mixture of GAGs that possess anticoagulant and anti-inflammatory properties.

A histamine-independent itch pathway is required for allergic ocular itch

Non-histaminergic TRPA1 neural pathway is required for the development of allergic ocular itch. Pharmacological inhibition of TRPA1 channel can lead to novel therapeutic strategies to treat ocular itch in severe allergic conjunctivitis.

Complex of tris(2-hydroxyethyl)amine and zinc bis(2-methylphenoxyacetate) as inhibitor of acid cholesterol esterase synthesis in blood platelets and mononuclear cells

Application of tris(2-hydroxyethyl)amine and zinc bis(2-methylphenoxyacetate) complex (zincatrane) in experimental atherosclerosis in rabbits leads to decrease in the levels of cholesterol and total lipids in blood platelets (Tc) and monocytes (Mc). The results of enzymatic analysis show that atherogenesis is accompanied by increased activity of acid cholesterol esterase by 66% in Mc and by 55% in Tc compared to standard values. Intramuscular administration of zincatrane at a dose of 10 mg kg−1 decreases the activity of acid cholesterol esterase by almost 35% in Mc and by 26% in Tc compared to the control group. It has been also revealed that zincatrane possesses heparin-like effect, which is expressed in increased thrombin clotting time, blood clotting time and plasma recalcification time as well as in the decreased thrombocyte aggregation. It is assumed that zincatrane stimulates production of endogenous heparin.

Mast Cells Increase Vascular Permeability by Heparin-Initiated Bradykinin Formation In Vivo

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability invivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

Endogenous heparin levels in the controlled asthmatic patient

Since heparin possesses anti-inflammatory properties, it is hypothesised that asthmatic patients have decreased levels of circulating heparin compared with healthy individuals. We compared endogenous heparin levels in controlled asthmatic patients (53 adults) from the Asthma Clinic at Johannesburg General Hospital with those of healthy controls (26 adults) from the general population. Heparin levels in the blood samples were tested using the Chromogenix Coatest Heparin kit. The blood of the patients contained significantly lower levels of endogenous heparin compared with that of the healthy individuals, indicating that the anti-inflammatory properties afforded by heparin are absent in these patients.

Locally available heparin modulates inflammatory cell recruitment in a manner independent of anticoagulant activity

Heparin is known to possess anti-inflammatory properties that in many cases appear to be separable from its anticoagulant activity. Mast cells, located in tissue, are the sole source of endogenous heparin, which may be involved in control of the inflammatory response. The majority of studies of the effects of heparin on the inflammatory response, carried out to date, have involved systemic administration and the potential influence of heparin in the site of inflammation has been less clear. In the present study, the effects of locally administered heparin and a non-anticoagulant derivative were investigated on leucocyte accumulation in the inflamed peritoneal cavity and leucocyte-endothelial interactions in the mesenteric microcirculation of the rat. Heparin and non-anticoagulant heparin inhibited the influx of neutrophils to the site of inflammation, as well as leucocyte rolling and adhesion in post-capillary venules. These effects were apparent only while heparin was present in the peritoneal cavity and plasma and may reflect, in part, an action on resident peritoneal cells, as the heparins tested were found also to inhibit the expression of endothelial adhesion molecules in response to products of activated mononuclear cells, in vitro. Our data show that locally administered heparin has anti-inflammatory effects in an in vivo model of peritoneal inflammation whilst present at the site of inflammation. These non-anticoagulant properties may involve interaction with cells in the site of inflammation, in addition to inhibiting cell adhesion at the level of the microcirculation.

Endogenous heparin activity is decreased in peripheral arterial occlusive disease

Naturally occurring heparin-like activity in the form of endogenous heparin and heparin sulfate proteoglycans has been shown in normal human plasma. Exogenous low-dose heparin improves pain-free walking distance and maximum walking distance in peripheral arterial occlusive disease (PAOD). Is reduced endogenous heparin activity responsible for some of the problems found in PAOD? This study compared heparin-like activity in patients with PAOD with that in healthy subjects and explored its relationship to disease severity. In part 1, native and heparinase-modified thromboelastography was performed on peripheral venous blood samples in three groups of patients to measure heparin-like anticoagulant activity. Group 1: 15 control subjects (median age, 60 years; range, 49-74 years; ankle-brachial pressure index [ABPI] >0.9); group 2: 14 patients with intermittent claudication (median age, 66 years; range, 56-80; ABPI, 0.69 [SD, 0.09]); group 3: 14 patients with rest pain (median age, 67.5 years; range, 54-84 years; ABPI, 0.45 [SD, 0.08]). In part 2, heparin equivalent to that in normal plasma was added to blood samples from 15 patients with short-distance claudication (n = 4) or rest pain (n = 11), and baseline (without heparinase) thromboelastography was performed to exclude lack of antithrombin as a cause of diminished heparin-like activity. In part 1, all patients with PAOD had a significant increase in coagulability compared with controls. Heparinase-modified thromboelastography in controls showed a significant decrease in the latent period between placing the sample in the analyser, where it is recalcified, to the initial fibrin formation (DeltaR time; P = .002) compared with native TEG, confirming endogenous heparin-like activity. Using DeltaR time as a measure of heparin-like activity, a significant reduction was found in patients with claudication (0.33 minutes; 95% confidence interval [CI], 0.004-0.65; P = .02) and in those with rest pain (0.25 minutes; 95% CI, -0.02 to 0.52; P = .02) compared with that in controls (0.78 minutes; 95% CI, 0.39-1.16). The DeltaR time also correlated with the ABPI (r = 0.35, P = .02), suggesting declining heparin-like activity with increasing ischemia. In part 2, exogenous heparin restored the thromboelastography in PAOD patients to normal, suggesting that lack of endogenous heparin-like compounds rather than reduced antithrombin levels was responsible for changes in coagulation. Patients with PAOD have reduced endogenous heparin-like activity that correlates with disease severity.

Inhibition of Heparinase I by Defibrotide: Potential Clinical Implications

Defibrotide is an anti-ischemic/antithrombotic agent used to treat transplantation associated vascular complications. Defibrotide releases endogenous antithrombotic mediators. Co-administration of defibrotide with heparin augments the effect of heparin and increases its biologic half-life. The effects of defibrotide and its fractions on heparinase I mediated digestion of heparin were investigated. A fixed concentration of heparin was subjected to Flavobacterium heparinicum heparinase I in the presence of graded amounts of defibrotide/fractions (6.25 to 250 μg/ml). The extent of heparin digestion was determined using HPLC and computation of the oligosaccharide profiles. Heparinase I digested heparin (MW = 15.2 kDa) to a low molecular mass heparin (MW = 4.1 kDa). At concentrations >125 μg/ml, defibrotide produced an almost complete inhibition of digestion. The IC50 value for this reaction was 12.5 μg/ml. A molecular weight dependence on the inhibition of heparinase was noted. Studies in primates revealed that the anticoagulant effects of both unfractionated and low molecular mass heparins are potentiated by defibrotide as determined by AUC measurements of the circulating level of these agents. The potentiation of the anticoagulant effect of heparin in patients treated simultaneously with defibrotide may be due in part to the inhibition of endogenous heparin degrading enzymes. These interactions should be taken into account to optimize anticoagulant management where these drugs are used in a combination regimen.

Inhibition of Heparinase I by Defibrotide with Potential Clinical Implications.

Defibrotide represents a poly-deoxyribonucleotide derived antischemic and antithrombotic agent. Currently this agent is used for the management of transplantation associated with vascular complications. Defibrotide is a polyanionic electrolyte capable of releasing endogenous antithrombotic mediators such as (tissue factor pathway inhibitor (TFPI) and heparans. Co-administration of defibrotide with heparin has been shown to produce an augmentation of the effect of heparin and an increase in the biologic half-life. This may be due to the drug interactions or inhibition of endogenous heparin digesting enzyme by defibrotide. To test the hypothesis that defibrotide may inhibit heparin digesting enzymes such as heparinases (I–III), the effects of defibrotide were investigated on the digestion of unfractionated heparin. Unfractionated heparin was subjected to bacterial heparinase I (flavobacterion heparinicum) in an isolated biochemical systems where unfractionated heparin was used as a substrate at a fixed concentration of 750 μg/ml. Graded amounts of defibrotide in the range of 6.25–250 μg/ml were supplemented to this mixture. The degree of heparinase digestion of unfractionated heparin was determined by using high performance liquid chromatographic methods and computation of oligosaccharide profiles. Heparinase I was found to digest heparin (MW=15.2kDa) converting it to low molecular mass heparins (MW=4.1kDa). At concentrations of >125 μg/ml defibrotide produced an almost complete inhibition of heparinase I digestion. This inhibition of heparinase digestion was dependent on defibrotide concentration and the IC50 was found to be 12.5 μg/ml. These results suggest that polyelectrolytes such as defibrotide are capable of inhibiting heparin/heparan digestion enzymes. The observed potentiation of anticoagulant effect of heparin in patients treated simultaneously with defibrotide may partially be due to the inhibition of endogenous heparin digesting enzymes. Subsequent studies in primates have revealed that the anticoagulant effects of both unfractionated heparin and low-molecular mass heparins are potentiated by defibrotide as determined by AUC measurements for the circulating level of these agents. Thus, these interactions should be taken into account to optimize anticoagulant management where these drugs are used in a combination regimen.

Etiology of Senile Osteoporosis

Osteoporosis is a major health problem characterized by compromised bone strength predisposing patients to an increased risk of fracture. It may cause morbidity and mortality in elderly men and women. The etiologic factors that lead to senile osteoporosis still are unclear. Based on the available experimental and clinical evidence, we update our earlier proposal that zinc deficiency plays a role in the pathogenesis of senile osteoporosis. However, the mechanism of zinc deficiency in osteoporosis remains unclear. Zinc deficiency may lead to the increase of endogenous heparin probably causing degranulation of mast cells and release of endogenous heparin, and an increase in the bone-resorbing effect of prostaglandin E2. Endogenous heparin and prostaglandin E2 are probably cofactors of parathyroid hormone and may have a role in the pathogenesis of senile osteoporosis enhancing the action of parathyroid hormone. Therefore, zinc replacement by dietary zinc supplementation might be valuable to prevent or treat senile osteoporosis. Additional studies are necessary for the evaluation of the possible role of zinc in the etiology of senile osteoporosis. Level V (expert opinion). See the Guidelines for Authors for a complete description of the levels of evidence.

Mo-P2:168 Endogenous heparin — A protective marker in men with myocardial infarction

Mo-P2:168 Endogenous heparin — A protective marker in men with myocardial infarction

Dyslipidemia, increased lipolysis, and depleted heparin system in insulin resistance

The purpose of this investigation was to make a comprehensive study of intravascular lipofysis, including extrahepatic lipoprotein lipase and hepatic triglyceride lipase in the serum, and the responsiveness of the heparin system in insulin resistance. Patients with arterial hypertension (n = 14), type 2 diabetes mellitus (n = 16), or metabolic syndrome (n = 10) were found to have higher serum lipolytic activity, triglyceridemia, lipidemia (in type 2 diabetes mellitus and metabolic syndrome) and hyperglycemia (in type 2 diabetes mellitus) than controls (10 healthy donors). Excessive blood intracellular reserve heparin secretion-induced release of extrahepatic lipoprotein lipase and hepatic triglyceride lipase into the vascular bed as a prerequisite for increasing the baseline level of lipolytic activity in insulin resistance pathology is discussed. The effects ofheparin (50 U/ml of serum) and insulin (2 U/ml of serum) on total lipoprotein lipase activity were not revealed in vitro. It is suggested that enhanced extracellular lipofysis and depleted endogenous heparin system result in the body's insulin resistance.

Proteolysis of Human Plasma Reveals the Presence of Complexes Formed by Endogenous Heparin and Peptides That Stimulate Angiogenesis

Human endogenous plasma heparin associates with proteins that mask its anticoagulant activity. This association persists after exhaustive proteolysis of plasma, and resulting peptide/heparin complexes have no anticoagulant activity. Looking for functions other than inhibition of coagulation, we considered that commercial preparations of heparin from bovine or porcine sources show alternative effects on angiogenesis, either stimulating or inhibiting the process. However, the effects of endogenous human heparin on angiogenesis are unknown. In this study, the fraction of plasma containing endogenous heparin was prepared by means of exhaustive proteolysis, either in the presence or in the absence of 35S-labeled heparin. Plasma from healthy donors was digested and the supernatant was precipitated with 66% ethanol, dialyzed, and submitted to basic and acidic ion-exchange chromatography. 35S-Labeled heparin as well as endogenous heparin bound plasmatic peptides, forming acidic, basic, and neutral complexes. Binding of peptides, eluting from both resins, impaired migration of heparin on cellulose acetate electrophoresis. Endogenous neutral complexes (i.e., those formed by human endogenous plasma heparin and peptides) were tested for angiogenic activity in chick embryo chorioallantoic membrane assay. Bovine heparin induced a moderate angiogenic response. Neutral complexes of human endogenous plasma heparin and basic plasma peptides induced a very strong angiogenic response. Treatment of neutral complexes with nitrous acid, which degrades heparin, abolished the angiogenic effect, thus demonstrating that it was due to the presence of heparin. These results demonstrate that proteolysis of human plasma generates angiogenic peptide/heparin complexes.