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We have demonstrated that 125I unfractionated heparin binds to cultured human skin fibroblasts with a Kd of 1.16 10(-8) M and is internalized partly. A low molecular weight heparin fraction (PK 10169) competed (50%) with 125I unfractionated heparin, but to a lesser extent than cold unfractionated heparin (90%). When the cell proliferation was induced by pure PDGF, heparin markedly potentiated the fibroblast growth. Similar stimulation was observed when the growth was induced by FGF or EGF. Low molecular weight heparin enhanced the fibroblast proliferation induced by PDGF but to a lesser extent than unfractionated heparin. Chondroitin sulfate has no effect. PDGF did not modify the heparin binding on fibroblast cultures either at 4 degrees C or 37 degrees C and did not alter the process of heparin internalization. PDGF binding to the cultured fibroblast (Kd 10.1 +/- 3.4 10(-10) M) was not modified by the presence of heparin when studied at 4 degrees C or 37 degrees C.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.

We analyzed the molecular weight distribution of (125I) heparin fractions bound or internalized by human endothelial cells, using gel permeation chromatography. Our results showed that high molecular weight heparin chains are selectively bound and internalized by endothelial cells. Endocytosis is followed by depolymerization of internalized heparin. Chloroquine prevented depolymerization of internalized heparin, indicating that lysosomal enzymes are involved in this process. Degradation of high molecular weight heparin chains by endothelial cells may contribute to the inactivation of the drug, especially as concerns the antifactor IIa activity.

The interaction of standard heparin and some low molecular weight heparin fragments (CY 222, mw 1,500-8,000 daltons) with human vascular endothelium in culture was studied using both 125I and 3H labeled ligands. A specific and saturable binding was shown for both labeled standard heparins. Two populations of binding sites for 3H-standard heparin could be distinguished: one of high affinity (KD = 0.12 microM), and another of lower affinity (KD = 1.37 microM). Total binding capacity was in the order of 10(7) molecules per cell. The same high level of affinity was calculated for unlabeled compounds from competition experiments with 125I-standard heparin. No other glycosaminoglycans, except a highly sulfated heparan (fraction IIA) could compete for heparin binding sites. A specific binding was also shown for 125I-CY 222. The affinity of unlabeled CY 222 was approximately ten times lower than that of unfractionated heparin. However, CY 222 could compete for approximatively 30% of standard heparin binding. Binding was not completely reversible. Even in the presence of a large excess of unlabeled compounds, a fraction of 25% of radioactive heparins remained bound to the endothelium. This fraction was three times lower if incubation was carried out at +4 degrees C, suggesting a possible incorporation of heparin into the endothelial cells.

We have previously shown that heparin was bound and internalized by cultured human endothelial cells. In this study, we have investigated the effect of heparin on endothelial cells growth. We found that heparin inhibited 3H-thymidine uptake as well as actual cell growth in a dose-dependent manner in the presence of low concentrations of human serum. Inhibition was maximal at 1% serum concentration and was abolished at 10%. Chasing experiments supported the role of membrane-bound heparin in this inhibition. Low molecular weight heparin fractions, or pentosan polysulfate, were equally effective in inhibiting 3H-thymidine uptake. On the other hand, the simultaneous addition of heparin and ECGS was synergic in stimulating 3H-thymidine uptake. These results suggest a modulatory role of heparin in endothelial cells growth.

Heparin is mainly known for its anticoagulant action, but today other biological effects are investigated. With the low molecular weight heparin fractions (LMWH), more homogenous, a more detailed study of the mechanism of action of heparins can be made. The anticoagulant action of heparin is mainly antithrombin III (AT III) dependent and the binding site of AT III on the heparin molecule has been recently identified. LMWH have a lower anticoagulant (anti-IIa) activity, and a relatively higher anti-Xa activity (ratio anti-Xa/anti-IIa = 5 to 10 for LMWH and 1 for standard heparin). The antithrombotic action of heparins is not strictly correlated to their anticoagulant activity. Other mechanisms of action, such as interactions with vascular endothelial cells and the fibrinolytic system may contribute to the antithrombotic action of heparins. New therapeutical possibilities are currently under investigation. Inhibition of vascular smooth muscle cells growth by heparin suggest a possible control of the atherosclerotic process by heparin. Moreover, heparin and its derivatives might be involved in the regulation of the cellular growth process.

Previous works have reported the controversial effects of heparin and steroids on angiogenesis. In this study, we investigated the effect of these compounds on human endothelial cell (EC) growth in vitro. An antiproliferative heparin activity was found in low human serum concentrations (2%). When EC were exposed to heparin (10(-6) M), their proliferation index was reduced in the presence of endothelial cell growth factor added 6 hours or more later. These results suggest that there is an intracellular effect of heparin which reduces 3H-methylthymidine uptake. Hydrocortisone acetate and tetrahydroS induced inhibition of EC growth in a dose-dependent manner. Steroids inhibited proliferation of EC in culture medium in the presence or the absence of growth factor and in different human serum concentrations. These results suggest a possible synergistic antiangiogenic action of heparin plus steroids.

Unfractionated heparin increased the binding of fibrinogen on ADP-treated platelets. The results varied according to both, the platelets of the donor and the kind of heparin preparation used. Beef lung heparin was more active than porcine intestinal mucosa heparin (p less than 0.02). A fraction of low molecular weight low sulfated heparin, did not significantly increase the binding of fibrinogen, except in one case for which the binding of fibrinogen to platelets was largely increased by standard heparin. On the contrary, pentosane polysulfate, a sulfated polysaccharide (of low molecular weight) significantly increased the binding of fibrinogen (p less than 0.01).

Cultured endothelial cells isolated from human umbilical vein bind heparin and heparin fragments. The binding capacity of endothelial cells for 35S-heparin was for 38% composed of high affinity binding sites (Kd = 11 X 10(-8) M) and for 62% of sites with much lower affinity (Kd = 14 X 10(-7) M). The affinity of unlabeled compounds for heparin binding sites was determined by competition with binding of 125I-heparin. I50 found for unlabeled heparin was 16 X 10(-8) M, which is in agreement with the Kd for binding of heparin to high affinity sites. PK 10169, a low molecular weight fragment of heparin, competed only at relatively high concentrations (I50 = 10(-5) M). Competition experiments with subfractions of PK 10169 showed that I50 was inversely correlated with molecular weight. Gelfiltration of 35S-heparin and 35S-PK 10169 before and after binding to endothelial cells demonstrated a selective binding of high molecular weight molecules from polydisperse heparin and PK 10169 preparations. Bound heparin and PK 10169 molecules were detached from the cell-surface by proteolytic treatment and tested for antifactor-Xa and antifactor-IIa activity. Released heparin is slightly more active in antifactor-Xa and antifactor-IIa activity than its parent preparation. Released PK 10169 was 4 fold more active in antifactor-Xa and 8 fold more active in antifactor-IIa assays than heparin.

Binding of heparin and low molecular weight heparin fragments (CY 222, Mr range 1500-8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [3H]heparin could be distinguished: one with high affinity (Kd = 0.12 microM) and another with lower affinity (Kd = 1.37 microM) and a relative large capacity of binding (1.16 X 10(7) molecules/cell) was calculated. The Kd for unlabeled heparin, as calculated from competition experiments, was 0.23 microM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 (Kd = 4.3 microM) from competition experiments with [125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25-30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4 degrees C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37 degrees C, but not at 4 degrees C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.

The availability of fibrinogen receptors on platelets after ADP stimulation, was investigated in order to analyze platelet hyperaggregability induced by heparin. Unfractionated heparin increased the binding of fibrinogen on ADP-treated platelets. The results varied according to both the donor platelets and the kind of heparin preparation used. Beef lung heparin was more active than porcine intestinal mucosa heparin (P less than 0.001). Low molecular weight (LMW) heparin fractions however did not significantly increase the binding of fibrinogen to ADP-treated platelets. The effect of standard heparin and LMW heparin on the aggregation of normal platelets induced by the addition of plasma from patients with heparin-induced immune thrombocytopenia was also studied. Concerning heparin-induced immune thrombocytopenia we have shown that the plasma cofactor present in some patient plasma may induce platelet aggregation both in the presence of standard-heparin and in the presence of LMW heparin fractions. Therefore, one has to be careful when replacing standard heparin by LMW heparin or heparinoids since each patient may react differently.

We have studied the binding of 125I-heparin to human omental microvascular endothelial cells (HOME cells) and investigated its effect on cell proliferation. At 20 degrees C, the binding reached a steady state from 4 hours onwards. Saturation of 125I-heparin binding occurred at 200 nM. Scatchard analysis indicated one class of binding sites (KD = 0.023 microM, 2 X 10(-6) sites/cell). Using fractionated heparin and other sulfated polysaccharides it was demonstrated that the binding was dependent on the charge and the molecular weight of the compounds. The binding was followed by the partial internalization of the bound ligand (23.8%). Heparin and Stipocus Japonicus mucopolysaccharide (SJAMP) inhibited the proliferation of exponentially growing HOME cells and human umbilical vein endothelial cells (HUVE cells). However, serum-deprived HOME cells were not inhibited by heparin.

In two patients treated by heparin for thrombosis, thrombocytopenia under 10 000/cumm occured accompanied with bleeding cud recurrent thrombosis. The demonstration in the patients' serum of a factor inducing platelet aggregation and serotonin release in the presence of heparin suggests that the thrombocytopenia could be of immuno-allergic origin. In both cases, the discontinuation of heparin resulted in clinical and biological improvement. The rare cases of heparin-induced thrombocytopenia reported in the literature are discussed.