Syndecan-4 regulates early neutrophil migration and pulmonary inflammation in response to lipopolysaccharide.

Proteoglycans (PGs) and their associated glycosaminoglycan side chains are effectors of inflammation, but little is known about changes to the composition of PGs in response to lung infection or injury. The goals of this study were to identify changes to heparan sulfate PGs in a mouse model of gram-negative pneumonia, to identify the Toll-like receptor adaptor molecules responsible for these changes, and to determine the role of the heparan sulfate PG in the innate immune response in the lungs. We treated mice with intratracheal LPS, a component of the cell wall of gram-negative bacteria, to model gram-negative pneumonia. Mice treated with intratracheal LPS had a rapid and selective increase in syndecan-4 mRNA that was regulated through MyD88-dependent mechanisms, whereas expression of several other PGs was not affected. To determine the role of syndecan-4 in the inflammatory response, we exposed mice deficient in syndecan-4 to LPS and found a significant increase in neutrophil numbers and amounts of CXC-chemokines and total protein in bronchoalveolar lavage fluid. In studies performed in vitro, macrophages and epithelial cells treated with LPS had increased expression of syndecan-4. Studies performed using BEAS-2B cells showed that pretreatment with heparin and syndecan-4 decreased the expression of CXCL8 mRNA in response to LPS and TNF-α. These findings indicate that the early inflammatory response to LPS involves marked up-regulation of syndecan-4, which functions to limit the extent of pulmonary inflammation and lung injury.

The severity of trauma determines the immune response to PF4/heparin and the frequency of heparin-induced thrombocytopenia.

Heparin can induce heparin-induced thrombocytopenia (HIT). The combined effect of type of surgery (major vs minor) and heparin on this prothrombotic immune reaction to platelet factor 4 (PF4)/heparin was analyzed. In a randomized, double-blind study, trauma patients receiving low-molecular-weight (LMWH) or unfractionated heparin (UFH) for thrombosis prophylaxis were assessed for PF4/heparin-antibody seroconversion, HIT, and thrombosis according to type of surgery. The risk for seroconversion was higher than major versus minor surgery odds ratio, 7.98 [95% confidence interval, 2.06-31.00], P = .003, controlled for potential confounders, as was the risk for HIT (2.2% [95% confidence interval, 0.3%-4.1%] vs 0.0%, P = .010). During LMWH compared with UFH thromboprophylaxis, HIT (1 of 298 vs 4 of 316; P = .370) and PF4/heparin seroconversion (1.7% vs 6.6%; P = .002) were less frequent, driven by differences in patients undergoing major surgery (incidence of HIT: LMWH 0.8% vs UFH 4.0%; P = .180; seroconversion rates: 4.0% vs 17.0%; P = .001). After minor surgery, no case of HIT occurred. The severity of trauma and the need for major surgery strongly influence the risk of an anti-PF4/heparin immune response, which is then increased by UFH. In major trauma certoparin may be safer than UFH because it induces HIT-antibody seroconversion, and the corresponding risk of HIT, less frequently.

Low molecular weight heparins differ substantially: impact on developing biosimilar drugs.

Generic drugs are an important component for meaningful health-care reform currently being debated in the United States. Aside from defining the period of drug exclusivity, however, there is a critical need to ensure that generics of biologic medicines (biosimilars) are safe and effective. For low molecular weight heparins (LMWHs), the standard of care for management of venous thromboembolism, their complex structure and polypharmacological actions make producing a generic LMWH more challenging than a generic small molecule medicine. Because biosimilar LMWHs will be used interchangeably with their branded product, inherent variability between products could lead to important differences in potency, safety, or effectiveness, including unanticipated immune responses. Awareness of the specific problems associated with biosimilar LMWH development led to new recommendations from several expert bodies. This article discusses the implications of these differences for the production of biosimilar LMWHs and provides recommendations to address the limitations in the pending U.S. Congress legislation, a well-intentioned undertaking but one that must preserve the health and welfare of citizens who require these critical care medications.

Heparin-induced thrombocytopenia in patients with Philadelphia-negative myeloproliferative disorders and unusual splanchnic or cerebral vein thrombosis.

BACKGROUND: Philadelphia-negative myeloproliferative disorders (Ph-MPD) are common causes of unusual splanchnic or cerebral vein thrombosis, which is treated with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). Heparin-induced thrombocytopenia (HIT) is a dangerous potential complication of this therapy, but it has rarely been reported in Ph-MPD. PATIENTS AND METHODS: We retrospectively reviewed clinical records of 29 patients with Ph-MPD who have been treated with UFH or LMWH for unusual splanchnic or cerebral vein thrombosis (3 cerebral sinus, 6 portal and 20 hepatic vein). The goal of the study was to determine the occurrence of new thrombotic events during heparin therapy secondary to HIT (HITT). RESULTS: During heparin therapy, 5 out of the 29 patients (17%) developed a new thrombotic episode (pulmonary embolism) with a high clinical probability of HIT based on the 4 T's score even though not all the patients developed 'true' thrombocytopenia. A diagnosis of HIT was established in 2 patients (6.8%) through the presence of heparin-related antibodies. CONCLUSIONS: Ph-MPD patients on heparin warrant careful monitoring and HIT has to be suspected whenever platelet counts drop or a new thrombosis is detectable.

Successful heparin desensitization after anaphylactic shock due to heparin.

A successful desensitization protocol in a patient with low molecular weight heparin induced anaphylactic reaction is being presented. A 72-years-old patient who was known to have multiple drug allergies and asthma was admitted with acute renal insufficiency. She had an anaphylactic reaction with a low molecular weight heparin during a hemodialysis session. Peritoneal dialysis was not feasible. Anticoagulation with warfarin was not considered appropriate; alternative anticoagulants were not available. Therefore a desensitization protocol was planned and applied, comprising of IV administration of diluted heparin by gradually increasing doses (0.1 to 5000 units), at 15 minute intervals, completing 8 hours before the procedure. By this way, IV heparin could be administered during the subsequent hemodialysis sessions with no reactions. The Naranjo probability scale revealed a probable adverse reaction associated with nadroparin for this patient. Anaphylactic reaction to low molecular weight heparins is reported rarely in the literature. To the best of our knowledge, this is the third case of successful heparin desensitization. When other anticoagulants are not available and anticoagulation is indispensible, heparin desensitization can be an option.

Close approximation of two platelet factor 4 tetramers by charge neutralization forms the antigens recognized by HIT antibodies.

OBJECTIVE: Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by antibodies that recognize positively charged platelet factor 4 (PF4), bound to the polyanion, heparin. The resulting immune complexes activate platelets. Unfractionated heparin (UFH) causes HIT more frequently than low-molecular-weight heparin (LMWH), whereas the smallest heparin-like molecule (the pentasaccharide, fondaparinux), induces anti-PF4/heparin antibodies as frequently as LMWH, but without exhibiting cross-reactivity with these antibodies. To better understand these findings, we analyzed the molecular structure of the complexes formed between PF4 and UFH, LMWH, or fondaparinux. METHODS AND RESULTS: By atomic force microscopy and photon correlation spectroscopy, we show that with any of the 3 polyanions, but in the order, UFH>LMWH>>fondaparinux--PF4 forms clusters in which PF4 tetramers become closely apposed, and to which anti-PF4/heparin antibodies bind. By immunoassay, HIT antibodies bind strongly to PF4/H/PF4 complexes, but only weakly to single PF4/heparin molecules. CONCLUSIONS: HIT antigens are formed when charge neutralization by polyanion allows positively charged PF4 tetramers to undergo close approximation. Whereas such a model could explain why all 3 polyanions form antibodies with similar specificities, the striking differences in the relative size and amount of complexes formed likely correspond to the observed differences in immunogenicity (UFH>LMWH approximately fondaparinux) and clinically relevant cross-reactivity (UFH>LMWH>>fondaparinux).

Allergy to heparins and anticoagulants with a similar pharmacological profile: an update.

Heparins are widely used as anticoagulants. Immunologically-mediated side effects raise the question as to whether other substances with heparin-like pharmacological effects can be safely applied. Hypersensitivity reactions to heparin consist of heparin-induced immune thrombocytopenia, allergic vasculitis, hypereosinophilia, immediate hypersensitivity as well as delayed-type skin reactions. Hypersensitivity to unfractionated and low-molecular-weight heparins and semisynthetic heparinoids is increasingly common, and the pathogenesis, however, is still not fully understood. Clinically, this phenomenon is of relevance because of its increasing incidence and the resulting therapeutic difficulties that arise because several cross-reactions between unfractionated and low-molecular-weight heparins as well as between various heparins and heparinoids have been observed. In some patients with cross-reactivity between various heparins and semisynthetic heparinoids, recombinant hirudins, may be safe and effective. Combined allergy to recombinant hirudins and heparins, however, has been reported. Therefore, there is an urgent need for therapeutic alternatives.

Anti Xa monitoring during treatment with low molecular weight heparin or danaparoid: inter-assay variability.

If laboratory monitoring of low molecular weight heparin (LMWH) therapy is required the test of choice is the anti Xa activity assay. The relationship between anti Xa results obtained using different techniques is unknown. The aim of the present study was to compare anti Xa results obtained with eight different commercially available anti Xa activity assays (five chromogenic and three clotting based assays) in samples from patients receiving either therapeutic or prophylactic LMWH (enoxaparin or dalteparin) or danaparoid. We have demonstrated that highly significant differences exist between results obtained using different techniques. The mean anti Xa activity in patients receiving treatment or prophylaxis with enoxaparin ranged from 0.28 to 0.64 iu/ml. A similar relationship was present in samples from patients treated with dalteparin, mean anti Xa results ranging from 0.43 to 0.69 iu/ml. The Heptest clotting assay as used here in combination with the Automated Coagulation Laboratory instrument, was associated with lower results than other clotting or chromogenic techniques. In patients receiving danaparoid for heparin induced thrombocytopaenia (HIT) mean results with three clotting based assays were 0.30 to 0.36 u/ml, compared to mean results of 0.47 to 0.65 u/ml for chromogenic assays. Our data clearly indicate that the selection of anti Xa assay method could influence patient management since the dose required to achieve the therapeutic range would differ according to the assay employed. This is particularly important since the frequency of haemorrhagic side effects has been shown by others to be dose dependent, irrespective of the concomitant anti Xa activity results. In danaparoid therapy the clotting assays studied here should not be employed for monitoring without a modified target range, unless it can be demonstrated that the higher doses required to achieve the therapeutic range are safe.

Heparin-induced thrombocytopenia: IgG binding to PF4-heparin complexes in the fluid phase and cross-reactivity with low molecular weight heparin and heparinoid.

Early diagnosis of heparin-induced thrombocytopenia (HIT) is essential to reduce morbidity and mortality. We report an enzyme immunoassay which detects the binding of HIT IgG to PF4-heparin in the fluid phase. Our fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. We were able to detect anti-PF4-heparin IgG in 26/28 (93%) HIT patients. We investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 23/26 (88%) of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration.

Cross-reactivity study of low molecular weight heparins and heparinoid in heparin-induced thrombocytopenia.

Previous studies suggested that the cross-reactivity rates of low molecular weight (LMW) heparins with the antibody in heparin-induced thrombocytopenia (HIT) are more than 80%, whilst that of the LMW heparinoid (Orgaran) is relatively low at about 10%. These earlier studies were limited either in the number of patients studied, or in investigating only a single drug. They were also inadequate due to non-standardisation of testing conditions. This study compares three LMW heparins (Fragmin, Clexane, Fraxiparin) and a heparinoid (Orgaran) in their cross-reactivity rates with the HIT antibody. The sera of 45 HIT patients were tested using platelet aggregometry under standardised conditions. The cross-reactivity rates are: 7% (Orgaran), 89% (Fragmin), 83% (Clexane) and 86% (Fraxiparin). Although there are no controlled trials to determine the in vivo cross-reactivity rates of LMW heparins and heparinoid, the low in vitro cross-reactivity rate of the latter favours its use in HIT. Nevertheless, the therapeutic use of these drugs in HIT should be preceded by exclusion of in vitro cross-reactivity.

Differential effects of clivarin and heparin in patients undergoing hip and knee surgery for the generation of anti-heparin-platelet factor 4 antibodies.

The pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated via a heterogeneous group of heparin(s)-platelet factor 4 (H-PF4) complexes bound to their antibodies. These anti-H-PF4 (AHPF4) antibodies that are capable of binding to the FcgammaRIIA receptor [cluster of differentiation (CD) 32] on platelets, resulting in platelet activation, widely vary in their specific activities as platelet activation (functionality). Predisposing factors related to specific pathologic conditions may also contribute to the generation of these antibodies and their relative functionality during HIT syndrome. To understand this phenomenon, a sub-study was carried out in patients undergoing elective total hip and knee replacement surgery (ECHOS Study) and who were treated with unfractionated heparin (UFH) and a low-molecular-weight heparin (LMWH; Clivarin). Approximately 600 patients per arm [UFH=7,500 anti-Xa U twice a day (b.i.d.) subcutaneous (s.c.) and clivarin=4200 U once daily (o.d.) s.c.], age >40 years, received prophylactic treatment for a minimum of 11-14 days. Plasma samples were collected at pre-dose, days 2-4, days 11-14 and at follow-up 6-8 weeks after discharge and were analyzed for AHPF4 antibody titers. Functionality of the enzyme-linked immunosorbant assay (ELISA)-positive AHPF4 antibodies to cause platelet activation was tested by 14C-serotonin release assay (SRA). Both UFH and clivarin treatments in orthopedic surgical patients resulted in a progressive generation of AHPF4 antibodies. The relative prevalence/functionality of AHPF4 antibodies in clivarin arm was markedly lower (two- to threefold, p<0.001) as compared to UFH at each time point. Most of the samples in clivarin group were found to be SRA negative, suggesting the presence of AHPF4 antibodies that did not activate platelets (nonfunctional). Within the UFH arm, the relative prevalence/functionality of AHPF4 antibodies was much higher (p<0.002) in knee group compared to the corresponding hip group. This study, for the first time, reports on the elevated levels of AHPF4 antibodies generated by heparin associated with the pathogenesis of knee surgery. Clinical significance of the differential generation of HIT-associated antibodies remains unexplored at this time.

Comparing the antithrombotic efficacy of a humanized anti-factor IX(a) monoclonal antibody (SB 249417) to the low molecular weight heparin enoxaparin in a rat model of arterial thrombosis.

A humanized inhibitory anti-factor IX(a) antibody (SB 249417) has been compared to enoxaparin (Lovenox) in a rat model of arterial thrombosis. Pretreatment of rats with either SB 249417 (3.0 mg/kg, i. v.) or enoxaparin (30.0 mg/kg, i.v. or s.c.) resulted in comparable and significant reductions in thrombus formation. However, the efficacious dose of enoxaparin resulted in >30-fold increase in the aPTT over baseline, while the efficacious dose of SB 249417 prolonged the aPTT by only approximately 3-fold. Additionally, pretreatment with SB 249417 resulted in sustained blood flow and arterial patency throughout the experiment in >80% of rats treated. In contrast, <30% of rats pretreated with enoxaparin remained patent throughout the experiment. The data in this report indicate that the selective inhibition of factor IX(a) with the monoclonal antibody SB 249417 produces a superior antithrombotic profile to that of the low molecular weight heparin enoxaparin.

Type IV hypersensitivity to subcutaneous heparin: a new case.

A 71-year-old woman was seen for cutaneous lesions that appeared on her abdomen 15 days after the beginning of subcutaneous injection of nadroparin calcium (Fraxiparina), a low molecular-weight heparin (LMWH). The lesions were very pruriginous, measured 0.5-3 cm, and appeared at and around the point of Fraxiparina injection. Fraxiparina treatment was stopped and the lesions subsided spontaneously in one week. The patient had been treated with intravenous heparin (H) one year before. A 6 mm punch biopsy showed spongiosis and a mild dermal superficial perivascular infiltrate composed of lymphocytes and eosinophils. A challenge test with a therapeutic dose of Fraxiparina produced a lesion similar to those described above. Epicutaneous, prick and intradermal tests with undiluted samples of different H (both preservative-containing and preservative-free) were performed. Patch tests produced a mild erythematous reaction to all H at 48 hours and a (++) reaction at 96 hours. These reactions persisted for one week. Prick tests showed neither an immediate nor a delayed reaction. Intradermal tests with H did not produce an immediate reaction, but induced an infiltrated erythematous reaction at 48 hours that enlarged during the next 2 days and was transformed into pruritic plaques with vesicles. The lesions cleared in two weeks. Our findings confirm a delayed-type hypersensitivity to H and cross-reactivity between unfractioned and LMWH.

[Re-examination of the role of mast cells, mediators of inflammation and heparin]. / Réexamen du rôle des mastocytes, des médiateurs de l'inflammation et de l'héparine.

Mast cells contain proteoglycans, e.g. heparin, which manifest potent anti-inflammatory features. Mast cell degranulation could eventually result in self limiting inflammation with shortened course due to heparin release. The recent availability of low molecular weight heparins will allow for clinical studies in allergic/inflammatory syndromes, e.g. asthma.

[Heparin-induced thrombopenia: significance and difficulties of precise identification of the immunologic mechanism]. / Thrombopénies induites par l'héparine: intérêt et difficultés de l'identification précise d'un mécanisme immunologique.

Heparin-induced thrombocytopenia (HIT) is a drug induced immunohematologic adverse reaction which is a rare but potentially very severe accident. Its diagnosis is important for epidemiologic and drug surveillance studies and in order to decide the most appropriate treatment. Its importance is enhanced since there is no gold standard diagnostic criteria. In clinical practice the diagnosis is based on a group of criteria related to clinical events and laboratory tests. We have established a score based on anamnestic criteria which allowed us to evaluate and compare two different laboratory tests: a platelet aggregation test (PAT) and a test for the detection of heparin dependent antibodies (Heparin Platelet Induced Antibodies or HPIA). The functional test PAT which is commonly used in expert laboratories detects antibodies inducing platelet aggregation in the presence of heparin. The HPIA test more recently developed is an ELISA test which detects antibodies directed at heparin-platelet factor 4 complexes. The relative value of theses two methods for the diagnosis of HIT is not well documented. We have analysed the results of these two tests in 273 consecutive patients with a suspicion of HIT. The results were concordant in 70% of patients. In selecting the patients with the lowest and the highest probability of HIT according to the score, PAT was found a more sensitive and HPIA a more specific test than the other. At low probability PAT is more often positive than HPIA 18% and 9% respectively. No test is 100% reliable, the specificity being limited for both tests since in about 20% of cases one or both tests are negative contrasting with a highly probable HIT. In this last group of patients, PAT was more frequently positive (86%) than HPIA (72%). Both tests are negative in 6% of patients suggesting the existence of presently unknown antigenic targets. Considering a group of 19 patients with a high probability of HIT, we have found antibodies against IL-8 or NAP-2 in only 7 patients. The discrepancy between a HPIA positive and a PAT negative encountered in 8% of patients may be explained by the existence of IgA or IgM immunoglobulins since in contrast to IgG they are unable to promote platelet aggregation via the CD32 platelet membrane receptor. This work suggests than neither test is 100% reliable and that they play a complementary role in the diagnosis of HIT. The potential advantage of using both tests should be confirmed in complementary studies

[Delayed hypersensitivity to heparins and heparinoids]. / Hypersensibilité retardée aux héparines et heparinoïdes.

Delayed hypersensitivity to heparins and heparinoïd is a problem for prophylaxis of thrombo embolic diseases. The hirudins did not seem to have any cross-reactivity with the two others groups of anticoagulants. We present two clinical cases of delayed type reactions to heparins and heparinoïd and we reviewed the literature about adverse reactions to low molecular weight heparins and the alternative possibilities.

[Arterial thrombosis due to heparin induced thrombocytopenia]. / Arteriële trombose door heparine-geïnduceerde trombocytopenie.

BACKGROUND: Type II heparin induced thrombocytopenia (HIT) is a procoagulant disorder that is caused by IgG-antibodies against platelet factor 4 (PF4)-heparin (H) complex. Clotting tendency is also increased. This is characterized by a ≥ 50% decrease in platelet count between 5-10 days after exposure to unfractionated or low-molecular weight heparin. CASE DESCRIPTION: A 49-year-old woman presented with neurological symptoms and pain in her right hand shortly after hospitalisation in Spain. She had an ischaemic CVA and arterial perfusion difficulties in her right arm due to a large thrombus in the aortic arch and some of its branches. She was treated with thrombolytic therapy and dalteparin. Based on initially mild thrombocytopenia that progressed rapidly after admission and her 7-day exposure to enoxaparin during the previous hospital admission, we diagnosed heparin induced thrombocytopenia (HIT) with arterial thrombosis. CONCLUSION: It can be difficult to diagnose HIT. A clinical probability score based on clinical parameters and laboratory results is useful in this. Quick diagnosis and treatment are of great importance because of the high risk of complications.