Introduction: Bovine and ovine mucosa represent alternate anticoagulants to porcine mucosa for production of unfractionated heparin (UFH). Standardized heparins from various sources can be blended and potency adjusted, blended heparins exhibit comparable effects as single-sourced porcine UFH. This study evaluated the pharmacologic profile of blended heparin and compared their activities to that of single sourced porcine, ovine, and bovine heparins. Methods: The anticoagulant effects of gravimetric and potency-adjusted heparins were evaluated with aPTT, TT, anti-Xa, anti-IIa, ACT, and TGA studies. Protamine sulfate studies were used for neutralization potential of each of the individual heparins. Results: The potency-adjusted heparins demonstrated comparable aPTT, TT, anti-Xa, anti-IIa, and ACT values at all concentrations (U/mL). However, in gravimetric studies, bovine heparin consistently showed lower values with the exception of thrombin generation inhibition studies. The protamine sulfate neutralization studies demonstrated complete neutralization at all concentrations for the potency-adjusted heparins. However, at gravimetric concentrations, minor differences were noted in the neutralization profile in each of these heparins. Conclusion: These studies support the hypothesis that blended heparin from bovine, ovine, and porcine tissue, when standardized in unit-equivalent proportions, exhibits a comparable anticoagulant profile to the single species derived heparins.

Patients with mechanical heart valves (MHV) have an increased risk of thromboembolic complications. Low molecular weight heparin (LMWH) and unfractionated heparin (UFH) are often recommended for bridging anticoagulation; however, it is not clear which strategy is more beneficial.

Heparin, a widely used anticoagulant, carries the risk of an antibody-mediated adverse drug reaction, heparin-induced thrombocytopenia (HIT). A subset of heparin-treated patients produces detectable levels of antibodies against complexes of heparin bound to circulating platelet factor 4 (PF4). Using a genome-wide association study (GWAS) approach, we aimed to identify genetic variants associated with anti-PF4/heparin antibodies that account for the variable antibody response seen in HIT. We performed a GWAS on anti-PF4/heparin antibody levels determined via polyclonal enzyme-linked immunosorbent assays. Our discovery cohort (n = 4237) and replication cohort (n = 807) constituted patients with European ancestry and clinical suspicion of HIT, with cases confirmed via functional assay. Genome-wide significance was considered at α = 5 × 10-8. No variants were significantly associated with anti-PF4/heparin antibody levels in the discovery cohort at a genome-wide significant level. Secondary GWAS analyses included the identification of variants with suggestive associations in the discovery cohort (α = 1 × 10-4). The top variant in both cohorts was rs1555175145 (discovery β = -0.112 [0.018], P = 2.50 × 10-5; replication β = -0.104 [0.051], P = .041). In gene set enrichment analysis, 3 gene sets reached false discovery rate-adjusted significance (q < 0.05) in both discovery and replication cohorts: "Leukocyte Transendothelial Migration," "Innate Immune Response," and "Lyase Activity." Our results indicate that genomic variation is not significantly associated with anti-PF4/heparin antibody levels. Given our power to identify variants with moderate frequencies and effect sizes, this evidence suggests genetic variation is not a primary driver of variable antibody response in heparin-treated patients with European ancestry.

Thrombosis in COVID-19 worsens mortality. In our study, we sought to investigate how the dose and type of anticoagulation (AC) can influence patient outcomes.

We evaluated the effects of T helper cell differentiation in a mite-allergic animal model treated with inhaled heparins of different molecular weight.

Tumors are angiogenesis dependent and vascular endothelial growth factor-A (VEGF-A), a heparin-binding protein, is a key angiogenic factor. As chemotherapy and co-treatment with anticoagulant low-molecular-weight heparin (LMWH) are common in cancer patients, we investigated whether angiogenesis in vivo mediated by VEGF-A is modulated by metronomic-type treatment with: (i) the LMWH dalteparin; (ii) low-dosage cytostatic epirubicin; or (iii) a combination of these two drugs. Using the quantitative rat mesentery angiogenesis assay, in which angiogenesis was induced by intraperitoneal injection of very low doses of VEGF, dalteparin sodium (Fragmin(®) ) and epirubicin (Farmorubicin(®) ) were administered separately or in combination by continuous subcutaneous infusion at a constant rate for 14 consecutive days. Dalteparin was administered at 27, 80, or 240 IU/kg/day, i.e., doses that reflect the clinical usage of this drug, while epirubicin was given at the well-tolerated dosage of 0.4 mg/kg/day. While dalteparin significantly stimulated angiogenesis in an inversely dose-dependent manner, epirubicin did not significantly affect angiogenesis. However, concurrent treatment with dalteparin and epirubicin significantly inhibited angiogenesis. The effect of dalteparin is the first demonstration of a proangiogenic effect of any LMWH in vivo. The fact that co-treatment with dalteparin and epirubicin significantly inhibited angiogenesis suggests a complex drug effect.

Intravenous unfractionated heparin (UFH) remains one of the most commonly used anticoagulants in the hospital setting. The optimal protocol for initiation and maintenance of UFH has been difficult to determine. Over the past two decades, weight-based nomogram protocols have gained favor. Herein, we present a retrospective study of 377 patients at a single tertiary academic center treated with low intensity (LI) and standard intensity (SI) UFH protocols for therapeutic anticoagulation. UFH levels are measured by anti-Xa assay activity with therapeutic levels of 0.30 to 0.70 IU/mL for SI and 0.25 to 0.35 IU/mL for LI.  Patients treated on the LI protocol were more likely to have had a previous history of bleeding and lower baseline hemoglobin. Incidence of new or worsening thrombus while on UFH was comparable between both protocols (odds ratio (OR) 0.93, 95% confidence interval (CI) 0.29-2.98, p=0.899). Patients on LI protocol had higher incidence of bleeding while on UFH (OR 1.21, 95% CI 0.51-2.89, p=0.667). Our study thus suggests that the LI protocol may have comparable efficacy to the SI protocol in treating venous thromboembolism (VTE) and that target anti-Xa levels of 0.25 to 0.35 IU/mL may be more optimal in high-risk patients.

Heparin and heparan sulfate (HS) are highly sulfated polysaccharides covalently bound to cell surface proteins, which directly interact with many extracellular proteins, including the transforming growth factor-β (TGFβ) family ligand antagonist, follistatin 288 (FS288). Follistatin neutralizes the TGFβ ligands, myostatin and activin A, by forming a nearly irreversible non-signaling complex by surrounding the ligand and preventing interaction with TGFβ receptors. The FS288-ligand complex has higher affinity than unbound FS288 for heparin/HS, which accelerates ligand internalization and lysosomal degradation; however, limited information is available for how FS288 interactions with heparin affect ligand binding. Using surface plasmon resonance (SPR) we show that preincubation of FS288 with heparin/HS significantly decreased the association kinetics for both myostatin and activin A with seemingly no effect on the dissociation rate. This observation is dependent on the heparin/HS chain length where small chain lengths less than degree of polymerization 10 (dp10) did not alter association rates but chain lengths >dp10 decreased association rates. In an attempt to understand the mechanism for this observation, we uncovered that heparin induced dimerization of follistatin. Consistent with our SPR results, we found that dimerization only occurs with heparin molecules >dp10. Small-angle X-ray scattering of the FS288 heparin complex supports that FS288 adopts a dimeric configuration that is similar to the FS288 dimer in the ligand-bound state. These results indicate that heparin mediates dimerization of FS288 in a chain-length-dependent manner that reduces the ligand association rate, but not the dissociation rate or antagonistic activity of FS288.

Studies using lipid infusions to raise fatty acid levels require heparin to release lipoprotein lipase (LPL), thus calling into question the appropriate control infusion for this type of study: saline alone or saline plus heparin. We aimed to evaluate whether the addition of heparin alone, in doses needed to release LPL, would alter circulating free fatty acids (FFAs) and/or affect gonadotropins.

A heparin-modified poly(N-isopropylacrylamide) (PIPAAm)-grafted surface bound with heparin-binding epidermal growth factor-like growth factor (HB-EGF) was able to culture hepatocytes maintaining high albumin secretion and high expression of hepatocyte-specific genes. However, the activity of HB-EGF on the surface and its binding effects on hepatocytes remain unclear. In this study, we investigated the temperature-dependent interactions of HB-EGF and EGF receptor (EGFR) with heparin-modified PIPAAm to evaluate the activity of HB-EGF on the surface. Quartz crystal microbalance (QCM) measurements revealed that the amounts of adsorbed HB-EGF on either the heparin-modified PIPAAm-grafted surface (heparin-IC1) or PIPAAm-grafted surfaces were almost the same regardless of swelling/deswelling of grafted PIPAAm chains. The heparin-IC1 surface bound to HB-EGF at 37 °C had the ability to bind to hepatocytes through specific affinity interaction with EGFR, whose activation was confirmed by western blotting. However, the physisorbed HB-EGF on the PIPAAm surface greatly diminished its activity. Taken together, the introduction of heparin into grafted PIPAAm chains on the surface plays a pivotal role in holding HB-EGF while preserving its activity. Hydration and swelling of surface-grafted PIPAAm chains at 20 °C greatly diminished the attachment of hepatocytes with HB-EGF bound to heparin-IC1, whereas hepatocytes were able to bind to HB-EGF bound to heparin-IC1 at 37 °C. Thus, the equilibrated affinity interaction between EGFRs and surface-bound HB-EGF was considered to be attenuated by steric hindrance due to hydration and/or swelling of grafted PIPAAm chains.

Several studies have confirmed the role of prophylactic low-molecular-weight heparin (LMWH) for venous thromboembolism (VTE) in neurosurgery; however, a paucity of literature has assessed its safety and efficacy versus prophylactic unfractionated heparin (UFH). The objective is to present a meta-analysis directly comparing prophylactic LMWH to UFH for the prevention of VTE in neurosurgery.

Two systematic reviews summarized the efficacy and safety of pharmacological prophylaxis for venous thromboembolism (VTE) after hepatic resection, but both lacked a discussion of the differences in the pharmacological prophylaxis of VTE in different ethnicities. Therefore, we aimed to evaluate the efficacy and safety of low-molecular-weight heparin (LMWH) or unfractionated heparin (UFH) for VTE prophylaxis in Asian and Caucasian patients who have undergone hepatic resection.

Oxytocin (OXT) is a small cyclic peptide that is administered to pregnant women to induce birth in cases where labour is prolonged. It has previously been observed that patients taking a low molecular weight heparin, dalteparin (DAL), and then prescribed, OXT experienced a swifter labour compared to women given OXT alone. Herein are described the interactions between OXT and a number of heparin-based oligosaccharides; DAL; fondaparinux (FP), which is a synthetic heparin oligosaccharide that represents the predominant antithrombin binding-site, and a family of chemically-derived heparin hexasaccharides. The latter oligosaccharides were chosen as they represent sequences found within the polysaccharide dalteparin. Furthermore, the carbohydrate chemical space was investigated by comparing the interaction between OXT and four chemically derivatived heparin hexasaccharides; I2S-A6S NS (DP6), I2OH-A6S NS (DP6-2OH, de-2-O-sulfated hexasaccharide), I2S-A6OH NS (DP6-6OH, de-6-O-sulfated hexasaccharide) and I2S-A6S NAc (DP6-NAc, de-N-sulfated hexasaccharide). The interactions between the peptide and oligosaccharides were studied using a series of 13C-1H and 15N-1H HSQC NMR experiments, at a range of temperatures. This approach allowed the binding epitopes of the peptide and oligosaccharides to be identified, highlighting that 6-O- and N-sulfation substituent groups of heparin are important for the interaction between the peptide and carbohydrate. This is an important observation as de-N-sulfation is a traditional method for decreasing the anticoagulation properties of heparin. Furthermore, low temperature experiments of the OXT : FP complex indicate that hydrogen-bonding is very important for the interaction between the peptide and oligosaccharide.

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse drug effect from unfractionated or low-molecular-weight heparin that results in thrombocytopenia and potentially catastrophic thrombosis. HIT occurs due to the development of platelet-activating antibodies against multimolecular complexes of platelet factor 4 and heparin. Given the frequency of thrombocytopenia and heparin use among hospitalized patients, calculation of the 4Ts Score is recommended to identify patients at increased likelihood of HIT and direct further evaluation. In patients with an intermediate or high probability 4Ts Score, an immunoassay and functional assay are recommended to confirm or refute the diagnosis of HIT. Heparin avoidance and initiation of nonheparin anticoagulation are the mainstays of acute HIT management. In this illustrated review, we provide visual summaries of the diagnosis and management of HIT, highlighting connections between pathophysiology and clinical care as well as summarizing efforts in quality improvement in the field. We further emphasize common pitfalls and pearls in diagnosis and management to encourage evidence-based care. We include graphical representation of the unique challenges of HIT with cardiopulmonary bypass and also delineate autoimmune HIT and its subtypes.

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway. PKR is activated to undergo autophosphorylation upon binding to double-stranded RNAs or RNAs that contain duplex regions. Activated PKR phosphorylates the α subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis. PKR is also activated by heparin, a highly sulfated glycosaminoglycan. We have used biophysical methods to define the mechanism of PKR activation by heparin. Heparins as short as hexasaccharide bind strongly to PKR and activate autophosphorylation. In contrast to double-stranded RNA, heparin activates PKR by binding to the kinase domain. Analytical ultracentrifugation measurements support a thermodynamic linkage model where heparin binding allosterically enhances PKR dimerization, thereby activating the kinase. These results indicate that PKR can be activated by small molecules and represents a viable target for the development of novel antiviral agents.

One hundred fifty-six cases of deep venous thrombosis were reviewed. A previous episode of thrombosis, seen in 47 percent of the patients, was the most common predisposing factor. Medical illness, operation and local trauma were other important predisposing factors. Treatment consisted of continuous intravenous heparin therapy monitored by the activated coagulation time of whole blood (ACT). Thirty-three patients with an average ACT greater than 180 seconds had a more rapid recovery and had no recurrent deep venous thrombosis, embolism or failure to improve. Recurrent thrombosis, embolism or failure to improve was seen in 7 of 65 patients with an average ACT below 180 seconds. Major bleeding, defined as life-threatening bleeding or bleeding requiring transfusion, was not increased when the ACT was over 180 seconds, although minor bleeding was increased. Bleeding was also increased in women, elderly and postoperative patients. We advocate the use of higher doses of heparin to treat most patients with deep venous thrombosis because of the more rapid and complete recovery.

Background. Neuropilin-1 (NRP-1) is a multidomain membrane protein with soluble isoforms interacting with a complex network of other membrane receptors, their respective ligands and heparan sulfate (HS). It is involved in the development of vasculature, neural patterning, immunological responses and pathological angiogenesis. Methods. We have characterised the binding of a Fc fusion of rat NRP-1 (Fc rNRP-1) and of a soluble isoform, corresponding to the first four extracellular domains of human NRP-1, shNRP-1, using optical biosensor-based binding assays with a library of heparin derivatives. Selective labelling of lysines protected upon heparin binding allowed their identification by mass spectrometry. Results. Fc rNRP-1 bound to heparin with high affinity (2.5 nM) and fast ka (9.8 × 10(6) M(-1)s(-1)). Unusually, NRP-1 bound both highly sulfated and completely desulfated stretches of heparin and exhibited a complex pattern of preferences for chemically modified heparins possessing one or two sulfate groups, e.g., it bound heparin with just a 6-O sulfate group better than heparin with any two of N-sulfate, 6-O sulfate and 2-O sulfate. Mass-spectrometry based mapping identified that, in addition to the expected the b1 domain, the a1, and c domains and the L2 linker were also involved in the interaction. In contrast, shNRP-1 bound heparin far more weakly. This could only be shown by affinity chromatography and by differential scanning fluorimetry. Discussion. The results suggest that the interaction of NRP-1 with HS is more complex than anticipated and involving a far greater extent of the protein than just the b1-b2 domains. NRP-1's preference for binding long saccharide structures suggests it has the potential to bind large segments of HS chains and so organise their local structure. In contrast, the four domain soluble isoform, shNRP-1 binds heparin weakly and so would be expected to diffuse away rapidly from the source cell.

This study was performed to evaluate the ability of N-(2-hydroxypropyl)-3-tri methylammonium chitosan chloride (HTCC), the cationically modified chitosan, to form biologically inactive complexes with unfractionated heparin and thereby blocking its anticoagulant activity. Experiments were carried out in rats in vivo and in vitro using the activated partial thromboplastin time (APTT) and prothrombin time (PT) tests for evaluation of heparin anticoagulant activity. For the first time we have found that HTCC effectively neutralizes anticoagulant action of heparin in rat blood in vitro as well as in rats in vivo. The effect of HTCC on suppression of heparin activity is dose-dependent and its efficacy can be comparable to that of protamine-the only agent used in clinic for heparin neutralization. HTCC administered i.v. alone had no direct effect on any of the coagulation tests used. The potential adverse effects of HTCC were further explored using rat experimental model of acute toxicity. When administered i.p. at high doses (250 and 500 mg/kg body weight), HTCC induced some significant dose-dependent structural abnormalities in the liver. However, when HTCC was administered at low doses, comparable to those used for neutralization of anticoagulant effect of heparin, no histopathological abnormalities in liver were observed.

The aim of this simulation is to educate emergency medicine interns, residents and advanced providers on the recognition, diagnosis, and management of heparin-induced thrombocytopenia (HIT). Communication, teamwork, and crisis resource management are incorporated into the case.